Therapeutic human anti-IL-1R1 monoclonal antibody

ABSTRACT

Antibodies that interact with interleukin-1 receptor type 1 (IL-1R1) are described. Methods of treating IL-1 mediated diseases by administering a pharmaceutically effective amount of antibodies to IL-1R1 are described. Methods of detecting the amount of IL-1R1 in a sample using antibodies to IL-1R1 are described.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/961,537, filed Aug. 7, 2013, now U.S. Pat. No. 8,710,203, which is acontinuation of and claims priority to U.S. application Ser. No.13/450,126, filed Apr. 18, 2012, now U.S. Pat. No. 8,518,407, which is acontinuation of U.S. application Ser. No. 12/210,313, filed Sep. 15,2008, now U.S. Pat. No. 8,236,559, which is a continuation of and claimspriority to U.S. non-provisional application Ser. No. 10/656,769, filedSep. 5, 2003, now U.S. Pat. No. 7,438,910, which claims priority to U.S.provisional application Ser. No. 60/408,719 filed Sep. 6, 2002, thedisclosures of each of which are incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to antibodies that bind interleukin-1 receptortype 1 (IL-1R1) protein. Compositions, particularly pharmaceuticalcompositions and methods for treating of IL-1 mediated diseases, such asrheumatoid arthritis, osteoarthritis, and other inflammatory conditions,are also provided.

SEQUENCE LISTING

The instant application contains a sequence listing which has beensubmitted via EFS-Web and is incorporated herein by reference in itsentirety. Said ASCII copy, created on Mar. 31, 2014, is named102728-0041-105_Seq.text, and is 117,691 bytes in size.

BACKGROUND OF THE INVENTION Antibody Development

Inflammation is the body's response to injuries resulting frommechanical damage, infection, or antigenic stimulation. Inflammatoryreactions often are expressed pathologically. Such conditions arise whenthe inflammation is expressed in an exaggerated manner, isinappropriately stimulated, or persists after the injurious agent isremoved.

The inflammatory response is mediated, inter alia, by cytokines. One ofthe most potent inflammatory cytokines yet discovered is interleukin-1(IL-1). An increase in IL-1 signaling causes persistent inflammationassociated with several diseases, and IL-1 is thought to be a keymediator in many diseases and medical conditions. This cytokine ismanufactured primarily (though not exclusively) by cells of themacrophage/monocyte lineage and may be produced in two forms: IL-1 alpha(IL-1α) and IL-1 beta (IL-1β).

IL-1 stimulates cellular responses by interacting with a heterodimericreceptor complex comprised of two transmembrane proteins, IL-1 receptortype I (IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP). IL-1first binds to IL-1R1; IL-1RAcP is then recruited to this complex(Greenfeder et al., 1995, J. Biol. Chem. 270:13757-13765; Yoon andDinarello, 1998, J. Immunology 160:3170-3179; Cullinan et al., 1998, J.Immunology 161:5614-5620), followed by signal transduction resulting inthe induction of a cellular response.

Cell-based binding studies suggest that IL-1RAcP stabilizes the IL-1Rsignaling complex by slowing the ligand off-rate (Wesche et al., 1998,FEBS Letters 429:303-306). While the interaction of the IL-1 with IL-1Rhas been thoroughly characterized, the interaction of IL-1RAcP withligand-bound receptor remains poorly defined. Since IL-1RAcP has nosignificant affinity for either IL-1 or IL-1R1 alone, but high affinityfor the complex, it follows that novel binding sites for IL-1RAcP arecreated by the IL-1/IL-1R binding event, which may even includecontributions from IL-1 residues (Ettorre et al., 1997, Eur. CytokineNetw. 8:161-171). Another molecule, IL-1 receptor antagonist (IL-1ra)competes with IL-1α and IL-1β for receptor binding but fails to recruitIL-1RAcP, resulting in an occupied but non-signaling receptor. IL-1activity can additionally be counterbalanced by IL-1R type II, a decoyreceptor that binds ligand but does not participate in signaling due toa truncated intracellular domain. IL-1ra and IL-1R type II act to reducethe severity and duration of IL-1 mediated inflammatory events (Wescheet al., 1998, FEBS Letters 429:303-306; Dripps et al., 1991, J. Biol.Chem. 266:10331-10336; Dripps et al., 1991, J. Biol. Chem.266:20331-20335).

Interleukin-1 inhibitors may be produced from any protein capable ofspecifically preventing activation of cellular receptors to IL-1, whichmay result from a number of mechanisms. Such mechanisms includedown-regulating IL-1 production, binding free IL-1, interfering withIL-1 binding to IL-1R, interfering with formation of the IL-1R-IL-1RAcPcomplex, or interfering with modulation of IL-1 signaling after bindingto its receptor. Classes of IL-1 inhibitors include:

-   -   interleukin-1 receptor antagonists such as IL-1ra, as described        below;    -   anti-IL-1R monoclonal antibodies (e.g., as disclosed in        published European Patent Application No. EP 623674, the        disclosure of which is hereby incorporated by reference);    -   IL-1 binding proteins such as soluble IL-1 receptors (e.g., as        disclosed in U.S. Pat. Nos. 5,492,888; 5,488,032; 5,464,937;        5,319,071; and 5,180,812; the disclosures of which are hereby        incorporated by reference);    -   anti-IL-1 monoclonal antibodies (e.g., as disclosed in        International Patent Application Publication Nos. WO 9501997, WO        9402627, WO 9006371, U.S. Pat. No. 4,935,343, EP 364778, EP        267611 and EP 220063, the disclosures of which are hereby        incorporated by reference);    -   IL-1 receptor accessory proteins and antibodies thereto (e.g.,        as disclosed in International Patent Application Publication        Nos. WO 96/23067 and WO 99/37773, the disclosure of which is        hereby incorporated by reference); and    -   inhibitors of interleukin-1β converting enzyme (ICE) or caspase        I (e.g., as disclosed in International Patent Application        Publication Nos. WO 99/46248, WO 99/47545, and WO 99/47154, the        disclosures of which are hereby incorporated by reference),        which can be used to inhibit IL-1β production and secretion;    -   interleukin-1β protease inhibitors; and    -   other compounds and proteins that block in vivo synthesis or        extracellular release of IL-1.

Exemplary IL-1 inhibitors are disclosed in the following references:U.S. Pat. Nos. 5,747,444; 5,359,032; 5,608,035; 5,843,905; 5,359,032;5,866,576; 5,869,660; 5,869,315; 5,872,095; 5,955,480; and 5,965,564;International Patent Application Publication Nos WO98/21957, WO96/09323,WO91/17184, WO96/40907, WO98/32733, WO98/42325, WO98/44940, WO98/47892,WO98/56377, WO99/03837. WO99/06426, WO99/06042, WO91/17249, WO98/32733,WO98/17661, WO97/08174, WO95/34326, WO99/36426, and WO99/36415; Europeanpatent applications Publication Nos. EP534978 and EP89479; and Frenchpatent application no. FR 2762514. The disclosures of all of theaforementioned references are hereby incorporated by reference.

Interleukin-1 receptor antagonist (IL-1ra) is a human protein that actsas a natural inhibitor of interleukin-1 and is a member of the IL-1family, which includes IL-1α and IL-1β. Preferred receptor antagonists(including IL-1ra and variants and derivatives thereof), as well asmethods of making and using thereof, are described in U.S. Pat. No.5,075,222; International Patent Application Publication Nos. WO91/08285; WO 91/17184; WO92/16221; WO93/21946; WO 94/06457; WO 94/21275;WO 94/21235; DE 4219626, WO 94/20517; WO 96/22793; WO 97/28828; and WO99/36541, Australian Patent Application No. AU9173636; and French PatentApplication No. FR2706772; the disclosures of which are incorporatedherein by reference. The proteins include glycosylated as well asnon-glycosylated forms of IL-1 receptor antagonists.

Specifically, three useful forms of IL-1ra and variants thereof aredisclosed and described in U.S. Pat. No. 5,075,222 (“the '222 patent”).IL-1raα is characterized by SDS-PAGE as a 22-23 kD molecule having anapproximate isoelectric point of 4.8, eluting from a Mono Q FPLC columnat around 52 mM NaCl in Tris buffer, pH 7.6. IL-1raβ is characterized asa 22-23 kD protein, eluting from a Mono Q column at 48 mM NaCl. BothIL-1raα and IL-1raβ are glycosylated. IL-1rax is characterized as a 20kD protein, eluting from a Mono Q column at 48 mM NaCl, and isnon-glycosylated. The '222 patent also discloses methods for isolatingthe genes responsible for coding the inhibitors, cloning the gene insuitable vectors and cell types, and expressing the gene to produce theinhibitors. While effective, IL-1ra has a relatively short half-life. Incurrent use, IL-1ra is administered once a day. The art would thusbenefit from an antagonist of the IL-1 receptor with an appreciablylonger half-life.

Preventing IL-1 signaling by inhibiting IL-1 from binding the IL-1receptor is an attractive therapeutic approach for treating IL-1mediated diseases. There is a need in the art for clinically effectiveinhibitors of the IL-1 signaling pathway that may ameliorate the effectsof IL-1 mediated diseases and are suitable for delivery into humanpatients. A human antibody that blocks IL-1 signaling would beparticularly advantageous in fulfilling this need and would provide alonger half-life than currently available therapy.

SUMMARY OF THE INVENTION

The invention provides monoclonal antibodies that bind to interleukin-1receptor type I (IL-1R1). Preferably, the antibodies inhibit IL-1signaling by competing with IL-1β and IL-1α binding to IL-1R1. Alsoprovided by this invention are hybridoma cell lines that produce, andmost preferably, secrete into cell culture media the monoclonalantibodies of the invention. The antibodies of the inventionsuccessfully block IL-1 signaling in human cells and are useful therebyin treating patients with IL-1 mediated diseases. The invention furtherprovides fusion proteins comprising the sequence of an antibody Fcregion and one or more sequences selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQID NO: 38, and SEQ ID NO: 40. Such molecules can be prepared usingmethods as described, for example, in WO 00/24782, which is incorporatedby reference. Such molecules can be expressed, for example, in mammaliancells (e.g. Chinese Hamster Ovary cells) or bacterial cells (e.g. E.coli cells).

In certain aspects, the invention provides antibodies, preferablymonoclonal antibodies, most preferably human antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises anamino acid sequence as set forth in any of SEQ ID NO: 2, SEQ ID NO: 6,or SEQ ID NO: 8, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

The invention also provides antibodies, preferably monoclonalantibodies, most preferably human antibodies, comprising a heavy chainand a light chain, wherein the light chain comprises an amino acidsequence as set forth in SEQ ID NO: 4 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

In certain aspects, antibodies of the invention comprise a heavy chainand a light chain, wherein the variable region of the heavy chaincomprises an amino acid sequence as set forth in any of SEQ ID NO: 10,SEQ ID NO: 14, or SEQ ID NO: 16 or an antigen-binding or animmunologically functional immunoglobulin fragment thereof. In otheraspects, the light chain variable region comprises an amino acidsequence as set forth in any of SEQ ID NO: 12 or SEQ ID NO: 18, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof. In additional aspects, the heavy chain comprises an amino acidsequence as set forth in any of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ IDNO: 34, or SEQ ID NO: 36, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof. In still further aspects,the light chain comprises an amino acid sequence as set forth in any ofSEQ ID NO: 38 or SEQ ID NO: 40, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof. Suchantibody chains are useful in preparing antibodies that bindspecifically to IL-1R1 and also in preparation of bispecific antibodiesin which the resulting molecule binds to IL-1R1 and/or to another targetmolecule (e.g., TNF or a TNF receptor).

The invention also provides antibodies that bind specifically to IL-1R1,wherein the heavy chain comprises a heavy chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 10, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, and the light chain comprises a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 12, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, the invention also provides antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises a firstvariable region, and wherein the first variable region comprises asequence that has at least 90%, more preferably at least 95%, and mostpreferably about 99% identity to the amino acid sequence as set forth inSEQ ID NO: 10, and wherein the light chain comprises a second variableregion, and wherein the second variable region comprises a sequence thathas at least 90%, more preferably at least 95%, and most preferablyabout 99%, identity to the amino acid sequence as set forth in SEQ IDNO: 12, wherein the antibody interacts with IL-1R1.

The invention further provides antibodies that specifically bind toIL-1R1, wherein the heavy chain comprises a heavy chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 14, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, and the light chain comprises a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 12, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises a firstvariable region, and wherein the first variable region comprises asequence that has at least 90%, more preferably at least 95%, and mostpreferably about 99%, identity to the amino acid sequence as set forthin SEQ ID NO: 14, and wherein the light chain comprises a secondvariable region, and wherein the second variable region comprises asequence that has at least 90%, more preferably at least 95%, and mostpreferably about 99%, identity to the amino acid sequence as set forthin SEQ ID NO: 12, wherein the antibody interacts with IL-1R1.

The invention also provides antibodies that bind specifically to IL-1R1,wherein the heavy chain comprises a heavy chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 16, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, and the light chain comprises a light chain variable regioncomprising an amino acid sequence as set forth in SEQ ID NO: 18, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

In certain aspects, the invention provides antibodies, comprising aheavy chain and a light chain, wherein the heavy chain comprises a firstvariable region, and wherein the first variable region comprises asequence that has at least 90%, more preferably at least 95%, and mostpreferably about 99%, identity to the amino acid sequence as set forthin SEQ ID NO: 16, and wherein the light chain comprises a secondvariable region, and wherein the second variable region comprises anamino acid sequence that has at least 90%, more preferably at least 95%,and most preferably about 99%, identity to the amino acid sequence asset forth in SEQ ID NO: 18, wherein the antibody interacts with IL-1R1.

The invention also provides antibodies that bind specifically to IL-1R1,wherein the heavy chain comprises an amino acid sequence as set forth inSEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, or SEQ ID NO: 30, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof, and the light chaincomprises an amino acid sequence as set forth in SEQ ID NO: 38, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof.

The invention further provides antibodies that bind specifically toIL-1R1, wherein the heavy chain comprises an amino acid sequence as setforth in SEQ ID NO: 32, SEQ ID NO: 34, or SEQ ID NO: 36, or anantigen-binding or an immunologically functional immunoglobulin fragmentthereof, and the light chain comprises an amino acid sequence as setforth in SEQ ID NO: 40, or an antigen-binding or an immunologicallyfunctional immunoglobulin fragment thereof.

The invention also provides embodiments of all of the foregoing that aresingle chain antibodies, single chain Fv antibodies, Fab antibodies,Fab′ antibodies and (Fab′)₂ antibodies.

In particular aspects, the invention provides a light chain comprisingan amino acid sequence as set forth in any of SEQ ID NO: 38 or SEQ IDNO: 40, or an antigen-binding or an immunologically functionalimmunoglobulin fragment thereof.

In addition, the invention provides a heavy chain comprising an aminoacid sequence as set forth in any of SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32,SEQ ID NO: 34 or SEQ ID NO: 36, or an antigen-binding or animmunologically functional immunoglobulin fragment thereof.

The invention also relates to isolated human antibodies thatspecifically bind IL-1R1, wherein the antibody comprises: (a) humanheavy chain framework regions, a human heavy chain CDR1 region, a humanheavy chain CDR2 region, and a human heavy chain CDR3 region; and (b)human light chain framework regions, a human light chain CDR1 region, ahuman light chain CDR2 region, and a human light chain CDR3 region. Incertain aspects, the human heavy chain CDR1 region can be the heavychain CDR1 region of 26F5, 27F2, or 15C4 as shown in FIG. 10 and thehuman light chain CDR1 region can be the light chain CDR1 region of26F5, 27F2, or 15C4 as shown in FIG. 11. In other aspects, the humanheavy chain CDR2 region can be the heavy chain CDR2 region of 26F5,27F2, or 15C4 as shown in FIG. 10 and the human light chain CDR2 regioncan be the light chain CDR2 region of 26F5, 27F2, or 15C4 as shown inFIG. 11. In still other aspects, the human heavy chain CDR3 region isthe heavy chain CDR3 region of 26F5, 27F2, or 15C4 as shown in FIG. 10,and the human light chain CDR3 region is the light chain CDR3 region of26F5, 27F2, or 15C4 as shown in FIG. 11.

In addition, the invention provides an isolated human antibody thatspecifically binds to interleukin-1 receptor type 1 (IL-1R1),comprising: a human heavy chain CDR1 region, wherein the heavy chainCDR1 has the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62, or SEQID NO: 63; a human heavy chain CDR2 region, wherein the heavy chain CDR2has the amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65, or SEQ IDNO: 66; and/or a human heavy chain CDR3 region, wherein the heavy chainCDR3 has the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 68, or SEQID NO: 69.

The invention also provides an isolated human antibody that specificallybinds to interleukin-1 receptor type 1 (IL-1R), comprising: a humanlight chain CDR1 region, wherein the light chain CDR1 has the amino acidsequence of SEQ ID NO: 70 or SEQ ID NO: 71; a human heavy chain CDR2region, wherein the heavy chain CDR2 has the amino acid sequence of SEQID NO: 72 or SEQ ID NO: 73; and/or a human heavy chain CDR3 region,wherein the heavy chain CDR3 has the amino acid sequence of SEQ ID NO:74 or SEQ ID NO: 75.

In certain embodiments, the antibodies of the invention bind to thethird domain of IL-1R1, which is shown in FIG. 17. Preferably, theepitope for an antibody of the invention consists of the amino acidsequence YSV, which is referred to as Epitope 4 herein and shown in FIG.24. The invention further relates to fusion proteins and other moleculescapable of binding to Epitope 4 (together with the aforementionedantibodies, collectively referred to herein as “specific bindingpartners”), such as may be prepared using methods as described, forexample, in WO 00/24782, which is incorporated by reference. Suchmolecules can be expressed, for example, in mammalian cells (e.g.Chinese Hamster Ovary cells) or bacterial cells (e.g. E. coli cells).

Furthermore, the invention provides a method for epitope mapping of aselected antigen. In one aspect, the method comprises the steps of: (a)generating a set of fusion proteins, wherein each fusion proteincomprises (i) avidin and (ii) a fragment of the antigen; (b) screeningthe set of fusion proteins for binding to one or more specific bindingpartners for the antigen; (c) isolating the fusion proteins on a mediumcomprising biotin, whereby the avidin binds to the biotin; and (d)analyzing the fusion proteins bound by the specific binding partner orpartners to determine binding sites on the antigen for the specificbinding partner or partners. In a particular aspect, the specificbinding partners are antibodies.

In additional embodiments, the invention provides methods for treatingan IL-1 mediated disease, condition or disorder, comprising the step ofadministering a pharmaceutically effective amount of one or a pluralityof monoclonal antibodies of the invention or an antigen-binding or animmunologically functional immunoglobulin fragment thereof to anindividual in need thereof.

The invention also provides methods for detecting the level of IL-1R1 ina biological sample, comprising the step of contacting the sample with amonoclonal antibody of the invention or antigen-binding fragmentthereof. The anti-IL-1R antibodies of the invention may be employed inany known assay method, such as competitive binding assays, direct andindirect sandwich assays, immunoprecipitation assays and enzyme-linkedimmunosorbent assays (ELISA) (See, Sola, 1987, Monoclonal Antibodies: AManual of Techniques, pp. 147-158, CRC Press, Inc.) for the detectionand quantitation of IL-1R. The antibodies can bind IL-1R with anaffinity that is appropriate for the assay method being employed.

Specific preferred embodiments of the invention will become evident fromthe following more detailed description of certain preferred embodimentsand the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict a cDNA sequence (FIG. 1A) encoding a humananti-IL-1R1 antibody heavy chain IgG1 constant region (SEQ ID NO: 1) andthe amino acid sequence (FIG. 1B) of a human anti-IL-1R1 antibody heavychain IgG1 constant region (SEQ ID NO: 2).

FIGS. 2A-2B depict a cDNA sequence (FIG. 2A) encoding a humananti-IL-1R1 antibody kappa chain constant region (SEQ ID NO: 3) and theamino acid sequence (FIG. 2B) of a human anti-IL-1R1 antibody kappachain constant region (SEQ ID NO: 4).

FIGS. 3A-3B depict a cDNA sequence (FIG. 3A) encoding a human anti-IL-1Rantibody heavy chain IgG2 constant region (SEQ ID NO: 5) and the aminoacid sequence (FIG. 3B) of a human anti-IL-1R1 antibody heavy chain IgG2constant region (SEQ ID NO: 6).

FIGS. 4A-4B depict a cDNA sequence (FIG. 4A) encoding a humananti-IL-1R1 antibody heavy chain IgG4 constant region (SEQ ID NO: 7) andthe amino acid sequence (FIG. 4B) of a human anti-IL-1R1 antibody heavychain IgG4 constant region (SEQ ID NO: 8).

FIGS. 5A-5B depict a cDNA sequence (FIG. 5A) encoding the 26F5anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 9) and theamino acid sequence (FIG. 5B) of the 26F5 anti-IL-1R1 antibody heavychain variable region (SEQ ID NO: 10).

FIGS. 6A-6B depict a cDNA sequence (FIG. 6A) encoding the 26F5anti-IL-1R1 antibody kappa chain variable region (SEQ ID NO: 11) and theamino acid sequence (FIG. 6B) of the 26F5 anti-IL-1R1 antibody kappachain variable region (SEQ ID NO: 12).

FIGS. 7A-7B depict a cDNA sequence (FIG. 7A) encoding the 27F2anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 13) and theamino acid sequence (FIG. 7B) of the 27F2 anti-IL-1R1 antibody heavychain variable region (SEQ ID NO: 14).

FIGS. 8A-8B depict a cDNA sequence (FIG. 8A) encoding the 15C4anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 15) and theamino acid sequence (FIG. 8B) of the 15C4 anti-IL-1R1 antibody heavychain variable region (SEQ ID NO: 16).

FIGS. 9A-9B depict a cDNA sequence (FIG. 9A) encoding the 15C4anti-IL-1R1 antibody kappa chain variable region (SEQ ID NO: 17) and theamino acid sequence (FIG. 9B) of the 15C4 anti-IL-1R1 antibody kappachain variable region (SEQ ID NO: 18).

FIG. 10 shows an amino acid sequence alignment of heavy chains fromanti-IL-1R1 antibodies designated 15C4 (SEQ ID NO: 80), 27F2 (SEQ ID NO:82), and 26F5 (SEQ ID NO: 84). The complementarity determining regions(CDRs) are underlined. CDR1 for 26F5 is designated SEQ ID NO: 61; for27F2 is designated SEQ ID NO: 62; for 15C4 is designated SEQ ID NO: 63.CDR2 for 26F5 is designated SEQ ID NO: 64; for 27F2 is designated SEQ IDNO: 65; for 15C4 is designated SEQ ID NO: 66. CDR1 for 26F5 isdesignated SEQ ID NO: 67; for 27F2 is designated SEQ ID NO: 68; for 15C4is designated SEQ ID NO: 69.

FIG. 11 shows an amino acid sequence alignment of light chains fromanti-IL-R1-γ antibodies designated 15C4 (SEQ ID NO: 81), 27F2 (SEQ IDNO: 83), and 26F5 (SEQ ID NO: 83). CDR1 for 26F5/27F2 is designated SEQID NO: 70; for 15C4 is designated SEQ ID NO: 71. CDR2 for 26F5/27F2 isdesignated SEQ ID NO: 72; for 15C4 is designated SEQ ID NO: 73. CDR3 for26F5/27F2 is designated SEQ ID NO: 74; for 15C4 is designated SEQ ID NO:75.

FIG. 12 is a graph illustrating the inhibitory effect of anti-IL-1R1antibodies on IL-1R/IL-1β/IL-1RAcP complex formation.

FIG. 13 is a graph showing the inhibitory effect of an anti-IL-1R1monoclonal antibody as described herein and designated 15C4 onIL-1R/IL-1α/IL-1 RacP complex formation.

FIG. 14 is a graph representing the ability of anti-IL-1R1 antibodies toblock IL-1β binding while not significantly interfering with binding ofIL-1ra compared with IgG control.

FIG. 15A is a graph showing inhibition of IL-6 production in primaryhuman chondrocytes by anti-IL-1R1 antibodies identified herein anddesignated 15C4, 26F5, and 27F2 compared with IL-1ra.

FIG. 15B is a graph showing inhibition of IL-6 production in primaryhuman chondrocytes by IL-1ra and monoclonal antibodies 15C4 and 27F2compared with the class of monoclonal antibodies represented by 10H7 and24E12.

FIG. 16 is a graph showing inhibition of IL-6 production in human wholeblood by anti-IL-1R1 monoclonal antibodies designated 15C4, 26F5, and27F2 compared with IL-1ra.

FIG. 17 depicts human amino acid (SEQ ID NO: 76) and nucleotide (SEQ IDNO: 77) and rat nucleotide (SEQ ID NO: 78) and amino acid (SEQ ID NO:79) 3^(rd) domain IL-1R1 sequences. The numbered bars above the humansequence indicate the different sites mutated to construct the 15different mutated proteins. The rat residues introduced by mutation arelisted below the rat nucleic acid sequence.

FIG. 18 shows Western blot analysis demonstrating anti-IL-1R1 monoclonalantibody recognition of IL-1R1 mutants.

FIG. 19 is a drawing representing (I) activation of the IL-1 signalingpathway, which starts with binding of IL-1β to IL-1R1, and recruitmentof IL-1RacP, and three classes of anti-IL-1R1 antibodies: (II) 3^(rd)domain epitope IL-1 blockers, (II) 3^(rd) domain epitope RAcP blockers,and (IV) 1^(st)/2^(nd) domain epitope IL-1 blockers.

FIG. 20 depicts the crystal structure of 15C4 and 27F2 with mutation 10as described herein. The gray residues indicate the 15C4 and 27F2epitopes.

FIG. 21 depicts the 15C4 epitopes in the third domain of extracellularIL-1R1.

FIG. 22 depicts 24E12 epitopes in the third domain of extracellularIL-1R1.

FIG. 23 depicts the amino acid sequence (SEQ ID NO: 59) of theavidin-human IL-1R1-FLAG chimeric protein of the invention.

FIG. 24 depicts the amino acid sequence (SEQ ID NO: 60) of anavidin-cynomolgus IL-1R1-FLAG chimeric protein. The recombinant chickenavidin (italicized) is joined to the mature extracellular domain ofcynomolgus IL-1R1 (underlined, with C-terminal FLAG tag in bold) by a 6amino acid linker. Four amino acids from human IL-1R1 that wereintroduced alone and in combination into the cynomolgus sequence are inbold under the cynomolgus sequence. Epitope 4 is bold, italicized, andunderlined.

FIG. 25A shows a Western blot analysis of anti-human IL1-R1 antibody(anti-huIL1-R1) binding to Il-1R1. The * indicates that antibodies wereused at 5 μg/mL, whereas in the remainder antibodies were used at 1μg/mL.

FIG. 25B shows a summary of the densitometric analysis of a duplicateset of Western blot experiments.

FIG. 26 shows graphs representing the binding of anti-huIL1R1 antibodiesto avidin IL-1R1-FLAG proteins in a multiplexed bead-based bindingassay.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited in this application are expressly incorporated byreference herein for any purpose.

Definitions

A disease or medical condition is considered to be an “interleukin-1(IL-1) mediated disease” if the naturally-occurring orexperimentally-induced disease or medical condition is associated withelevated levels of IL-1 in bodily fluids or tissue or if cells ortissues taken from the body produce elevated levels of IL-1 in culture.Elevated levels of IL-1 can include, for example, levels that exceedthose normally found in a particular cell or tissue, or can be anydetectable level of IL-1 in a cell or tissue that normally does notexpress IL-1. In many cases, IL-1 mediated diseases are also recognizedby the following additional two conditions: (1) pathological findingsassociated with the disease or medical condition can be mimickedexperimentally in animals by administration of IL-1 or up-regulation ofexpression of IL-1; and (2) a pathology induced in experimental animalmodels of the disease or medical condition can be inhibited or abolishedby treatment with agents that inhibit the action of IL-1. In most IL-1mediated diseases at least two of the three conditions are met, and inmany IL-1 mediated diseases all three conditions are met.

A non-exclusive list of acute and chronic IL-1-mediated diseasesincludes but is not limited to the following: acute pancreatitis;amyelolateroschlerosis (ALS); Alzheimer's disease; cachexia/anorexia,including AIDS-induced cachexia; asthma and other pulmonary diseases;atherosclerosis; autoimmune vasculitis; chronic fatigue syndrome;Clostridium associated illnesses, including Clostridium-associateddiarrhea; coronary conditions and indications, including congestiveheart failure, coronary restenosis, myocardial infarction, myocardialdysfunction (e.g., related to sepsis), and coronary artery bypass graft;cancer, such as multiple myeloma and myelogenous (e.g., AML or CML) andother leukemias, as well as tumor metastasis; diabetes (e.g.,insulin-dependent diabetes); endometriosis; fever, fibromyalgia;glomerulonephritis; graft versus host disease/transplant rejection;hemorrhagic shock; hyperalgesia; inflammatory bowel disease;inflammatory conditions of a joint, including osteoarthritis, psoriaticarthritis and rheumatoid arthritis; inflammatory eye disease, as may beassociated with, e.g., corneal transplant; ischemia, including cerebralischemia (e.g., brain injury as a result of trauma, epilepsy, hemorrhageor stroke, each of which may lead to neurodegeneration); Kawasaki'sdisease; learning impairment; lung diseases (e.g., ARDS); multiplesclerosis; myopathies (e.g., muscle protein metabolism, especially insepsis); neurotoxicity (e.g., as induced by HIV); osteoporosis; pain,including cancer-related pain; Parkinson's disease; periodontal disease;pre-term labor; psoriasis; reperfusion injury; septic shock; sideeffects from radiation therapy; temporal mandibular joint disease; sleepdisturbance; uveitis; or an inflammatory condition resulting fromstrain, sprain, cartilage damage, trauma, orthopedic surgery, infectionor other disease processes. Methods of the invention for treating theseacute and chronic IL-1-mediated diseases, as well as other IL-1-mediatedconditions and diseases, are described below.

Conventional techniques may be used for preparing recombinant DNA,performing oligonucleotide synthesis, and practicing tissue culture andtransformation (e.g., electroporation, transfection or lipofection).Enzymatic reactions and purification techniques may be performedaccording to manufacturer's specifications or as commonly accomplishedin the art or as described herein. The foregoing techniques andprocedures may be generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclatureutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques may be used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “isolated polynucleotide” means that the subjectpolynucleotide, (1) is not associated (covalently or noncovalently) withall or a portion of other polynucleotides with which the subjectpolynucleotide is associated in nature, (2) is associated with amolecule with which it is not associated in nature, or (3) does notoccur in nature associated with any other polynucleotides. Such anisolated polynucleotide may be genomic DNA, cDNA, mRNA or other RNA, ofsynthetic origin, or any combination thereof.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldnormally be found, (2) is essentially free of other proteins from thesame source, e.g., from the same species, (3) is expressed by a cellfrom a different species, (4) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is associated in nature, (5) is not associated (bycovalent or noncovalent interaction) with portions of a protein withwhich the “isolated protein” is associated in nature, (6) is operablyassociated (by covalent or noncovalent interaction) with a polypeptidewith which it is not associated in nature, or (7) does not occur innature. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, orany combination thereof may encode such an isolated protein. Preferably,the isolated protein is substantially free from proteins or polypeptidesor other contaminants that are found in its natural environment thatwould interfere with its therapeutic, diagnostic, prophylactic, researchor other use.

The terms “polypeptide” or “protein” means one or more chains of aminoacids, wherein each chain comprises amino acids covalently linked bypeptide bonds, and wherein said polypeptide or protein can comprise aplurality of chains noncovalently and/or covalently linked together bypeptide bonds, having the sequence of native proteins, that is, proteinsproduced by naturally-occurring and specifically non-recombinant cells,or genetically-engineered or recombinant cells, and comprise moleculeshaving the amino acid sequence of the native protein, or moleculeshaving deletions from, additions to, and/or substitutions of one or moreamino acids of the native sequence. The terms “polypeptide” and“protein” specifically encompass anti-IL1-R1 antibodies, or sequencesthat have deletions from, additions to, and/or substitutions of one ormore amino acid of an anti-ILR-1R1 antibody. Thus, a “polypeptide” or a“protein” can comprising one (termed “a monomer”) or a plurality (termed“a multimer”) of amino acid chains.

The term “polypeptide fragment” refers to a polypeptide, which can bemonomeric or multimeric, that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion or substitutionof a naturally-occurring or recombinantly-produced polypeptide. Incertain embodiments, a polypeptide fragment can comprise an amino acidchain at least 5 to about 500 amino acids long. It will be appreciatedthat in certain embodiments, fragments are at least 5, 6, 8, 10, 14, 20,50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long.Particularly useful polypeptide fragments include functional domains,including binding domains. In the case of an anti-IL1-R1 antibody,useful fragments include, but are not limited to: a CDR region,especially a CDR3 region of the heavy or light chain; a variable domainof a heavy or light chain; a portion of an antibody chain or just itsvariable region including two CDRs; and the like.

The term “immunologically functional immunoglobulin fragment” as usedherein refers to a polypeptide fragment that contains at least thevariable domains of the immunoglobulin heavy and light chains. Animmunologically functional immunoglobulin fragment of the invention iscapable of binding to a ligand, preventing binding of the ligand to itsreceptor, interrupting the biological response resulting from ligandbinding to the receptor, or any combination thereof. Preferably, animmunologically functional immunoglobulin fragment of the inventionbinds specifically to IL-1R1.

The terms “naturally occurring” and “native” mean that the biologicalmaterials (molecules, sequences, protein complexes, cells, and the like)to which the terms are applied can be found in nature and are notmanipulated by man. For example, a polypeptide or polynucleotidesequence that is present in an organism (including viruses) that can beisolated from a source in nature and that has not been intentionallymodified by man is naturally occurring. Likewise, the terms“non-naturally occurring” or “non-native” refer to a material that isnot found in nature or that has been structurally modified orsynthesized by man.

The term “operably linked” means that the components to which the termis applied are in a relationship that allows them to carry out theirinherent functions under suitable conditions. For example, a controlsequence “operably linked” to a protein coding sequence is ligatedthereto so that expression of the protein coding sequence is achievedunder conditions compatible with the transcriptional activity of thecontrol sequences.

The term “control sequence” means that the subject polynucleotidesequence can effect expression and processing of coding sequences towhich it is ligated. The nature of such control sequences may dependupon the host organism. In particular embodiments, control sequences forprokaryotes may include a promoter, ribosomal binding site, andtranscription termination sequence. In other particular embodiments,control sequences for eukaryotes may include promoters comprising one ora plurality of recognition sites for transcription factors,transcription enhancer sequences, and transcription terminationsequence. In certain embodiments, “control sequences” can include leadersequences and/or fusion partner sequences.

The term “polynucleotide” means single-stranded or double-strandednucleic acid polymers of at least 10 bases in length. In certainembodiments, the nucleotides comprising the polynucleotide can beribonucleotides or deoxyribonucleotides or a modified form of eithertype of nucleotide. Said modifications include base modifications suchas bromouridine and inosine derivatives, ribose modifications such as2′,3′-dideoxyribose, and internucleotide linkage modifications such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate andphosphoroamidate. The term includes single and double stranded forms ofDNA.

The term “oligonucleotide” means a polynucleotide comprising a length of200 bases or fewer. In preferred embodiments, oligonucleotides are 10 to60 bases in length. In more preferred embodiments, oligonucleotides are12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length.Oligonucleotides may be single stranded or double stranded, e.g., foruse in the construction of a mutant gene. Oligonucleotides of theinvention may be sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” includes deoxyribonucleotidesand ribonucleotides. The term “modified nucleotides” includesnucleotides with modified or substituted sugar groups or modified orsubstituted bases. The term “oligonucleotide linkages” includes linkagessuch as phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See, e.g., LaPlanche et al. (1986),Nucl. Acids Res. 14:9081; Stec et al. (1984), J. Am. Chem. Soc.106:6077; Stein et al. (1988), Nucl. Acids Res. 16:3209; Zon et al.(1991), Anti-Cancer Drug Design 6:539; Zon et al. (1991),Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F.Eckstein, ed.), Oxford University Press, Oxford England; Stec et al.,U.S. Pat. No. 5,151,510; Uhlmann and Peyman (1990), Chemical Reviews90:543, the disclosures of which are hereby incorporated by referencefor any purpose. An oligonucleotide of the invention can include alabel, including a radiolabel, a fluorescent label, a hapten or anantigenic label, for detection assays.

The term “vector” means any molecule (e.g., nucleic acid, plasmid, orvirus) used to transfer coding information to a host cell.

The term “expression vector” or “expression construct” refers to avector that is suitable for transformation of a host cell and containsnucleic acid sequences that direct and/or control (in conjunction withthe host cell) expression of one or more heterologous coding regionsoperatively linked thereto. An expression construct may include, but isnot limited to, sequences that affect or control transcription,translation, and RNA splicing, if introns are present, of a codingregion operably linked thereto.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a selected gene of interest. The term includes the progeny ofthe parent cell, whether or not the progeny is identical in morphologyor in genetic make-up to the original parent cell, so long as theselected gene is present.

The term “transduction” means the transfer of genes from one bacteriumto another, usually by phage. “Transduction” also refers to theacquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, Id.; Davis et al., 1986, BasicMethods in Molecular Biology, Elsevier; and Chu et al., 1981, Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA. For example, a cell is transformed where itis genetically modified from its native state by transfection,transduction, or other techniques. Following transfection ortransduction, the transforming DNA may recombine with that of the cellby physically integrating into a chromosome of the cell, or may bemaintained transiently as an episomal element without being replicated,or may replicate independently as a plasmid. A cell is considered tohave been “stably transformed” when the transforming DNA is replicatedwith the division of the cell.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments,may have specific three-dimensional structural characteristics, and/orspecific charge characteristics. An epitope is a region of an antigenthat is bound by an antibody. In certain embodiments, an antibody issaid to specifically bind an antigen when it preferentially recognizesits target antigen in a complex mixture of proteins and/ormacromolecules. In preferred embodiments, an antibody is said tospecifically bind an antigen when the dissociation constant is less thanor equal to about 10 nM, more preferably when the dissociation constantis less than or equal to about 100 pM, and most preferably when thedissociation constant is less than or equal to about 10 pM.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by comparing the sequences thereof. In the art, “identity”also means the degree of sequence relatedness between nucleic acidmolecules or polypeptides, as the case may be, as determined by thematch between sequences of two or more nucleotides or two or more aminoacids. “Identity” measures the percentage of identical matches betweenthe smaller of two or more sequences with gap alignments (if any)addressed by a particular mathematical model or computer program (i.e.,“algorithms”).

The term “similarity” is used in the art with regard to a relatedconcept; in contrast to “identity,” however, “similarity” refers to ameasure of relatedness that includes both identical matches andconservative substitution matches. If two polypeptide sequences have,for example, 10/20 identical amino acids, and the remainder are allnon-conservative substitutions, then the percentage identity andsimilarity would both be 50%. If in the same example, there are fivemore positions where there are conservative substitutions, then thepercentage identity remains 50%, but the percentage similarity would be75% (15/20). Therefore, in cases where there are conservativesubstitutions, the percentage similarity between two polypeptides willbe higher than the percentage identity between those two polypeptides.

Identity and similarity of related nucleic acids and polypeptides can bereadily calculated by known methods. Such methods include, but are notlimited to, those described in Computational Molecular Biology, (Lesk,A. M., ed.), 1988, Oxford University Press, New York; Biocomputing:Informatics and Genome Projects, (Smith, D. W., ed.), 1993. AcademicPress, New York; Computer Analysis of Sequence Data, Part 1, (Griffin,A. M., and Griffin, H. G., eds.), 1994, Humana Press, New Jersey; vonHeinje, G., Sequence Analysis in Molecular Biology, 1987, AcademicPress; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.),1991, M. Stockton Press, New York; Carillo et al., 1988, SIAM J. AppliedMath. 48:1073; and Durbin et al., 1998, Biological Sequence Analysis,Cambridge University Press.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity aredescribed in publicly available computer programs. Preferred computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package, including GAP (Devereux etal., 1984, Nucl. Acid. Res. 12:387; Genetics Computer Group, Universityof Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul etal., 1990, J. Mol. Biol. 215:403-410). The BLASTX program is publiclyavailable from the National Center for Biotechnology Information (NCBI)and other sources (BLAST Manual. Altschul et al. NCB/NLM/NIH Bethesda,Md. 20894; Altschul et al., 1990, supra). The well-known Smith Watermanalgorithm may also be used to determine identity.

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, in certain embodiments, the selected alignmentmethod (GAP program) will result in an alignment that spans at least 50contiguous amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group,University of Wisconsin, Madison, Wis.), two polypeptides for which thepercentage sequence identity is to be determined are aligned for optimalmatching of their respective amino acids (the “matched span”, asdetermined by the algorithm). In certain embodiments, a gap openingpenalty (which is calculated as three-times the average diagonal; wherethe “average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually one-tenth of the gap openingpenalty), as well as a comparison matrix such as PAM250 or BLOSUM 62 areused in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see Dayhoff et al., 1978, Atlas of ProteinSequence and Structure 5:345-352 for the PAM 250 comparison matrix;Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for theBLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequencecomparison include the following:

-   -   Algorithm: Needleman et al. (1970), J. Mol. Biol. 48:443-453;    -   Comparison matrix: BLOSUM 62 from Henikoff et al. (1992), supra;    -   Gap Penalty: 12    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0        The GAP program may be useful with the above parameters. In        certain embodiments, the aforementioned parameters are the        default parameters for polypeptide comparisons (along with no        penalty for end gaps) using the GAP algorithm.

The term “naturally occurring,” as used to refer to amino acids, refersto the twenty conventional amino acids. See Immunology—A Synthesis, 2ndEdition, (E. S. Golub and D. R. Gren, eds.), Sinauer Associates:Sunderland, Mass. (1991), incorporated herein by reference for anypurpose.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compounds are termed “peptidemimetics” or “peptidomimetics”. See Fauchere (1986), Adv. Drug Res.15:29; Veber & Freidinger, 1985, TINS p. 392; and Evans et al. (1987),J. Med. Chem. 30:1229, which are incorporated herein by reference forany purpose. Such compounds are often developed with the aid ofcomputerized molecular modeling. Peptide mimetics that are structurallysimilar to therapeutically useful peptides may be used to produce asimilar therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm peptide or polypeptide (i.e., apeptide or polypeptide that has a biochemical property orpharmacological activity), such as human antibody, but have one or morepeptide linkages optionally replaced by a linkage selected from:—CH₂—NH—, —CH₂—S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—,—CH(OH)CH₂—, and —CH₂SO—, by methods well known in the art. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) maybe used in certain embodiments to generate more stable peptides. Inaddition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods known in the art (Rizo & Gierasch, 1992, Ann. Rev. Biochem.61:387, incorporated herein by reference for any purpose); for example,by adding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

“Antibody” or “antibody peptide(s)” refer to an intact antibody, or abinding fragment thereof that competes with the intact antibody forspecific binding and includes chimeric, humanized, fully human, andbispecific antibodies. In certain embodiments, binding fragments areproduced by recombinant DNA techniques. In additional embodiments,binding fragments are produced by enzymatic or chemical cleavage ofintact antibodies. Binding fragments include, but are not limited to,Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer specificityfor IL-1R1. A full-length heavy chain includes a variable region domain,V_(H), and three constant region domains, C_(H) ¹, C_(H) ², and C_(H) ³.The V_(H) domain is at the amino-terminus of the polypeptide, and theC_(H) ³ domain is at the carboxyl-terminus.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer specificityfor IL-1R1. A full-length light chain includes a variable region domain,V_(L), and a constant region domain. C_(L). Like the heavy chain, thevariable region domain of the light chain is at the amino-terminus ofthe polypeptide.

A “Fab fragment” is comprised of one light chain and the C_(H) ¹ andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H) ¹ and C_(H) ²domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H) ¹ andC_(H)2 domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and5,260,203, the disclosures of which are incorporated by reference forany purpose.

A “bivalent antibody” other than a “multispecific” or “multifunctional”antibody, in certain embodiments, is understood to comprise bindingsites having identical antigenic specificity.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol.79:315-321; Kostelny et al. (1992), J. Immunol. 148:1547-1553.

In assessing antibody binding and specificity according to theinvention, an antibody “substantially inhibits” adhesion of a ligand toa receptor when an excess of antibody reduces the quantity of receptorbound to counterreceptor by at least about 20%, 40%, 60%, 80%, 85%, ormore (as measured in an in vitro competitive binding assay).

The term “agent” means a chemical compound, a mixture of chemicalcompounds, a biological macromolecule, or an extract made frombiological materials.

The terms “label” or “labeled” refers to incorporation of a detectablemarker, e.g., by incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotin moieties that can be detected bymarked avidin (e.g., streptavidin preferably comprising a detectablemarker such as a fluorescent marker, a chemiluminescent marker or anenzymatic activity that can be detected by optical or colorimetricmethods). In certain embodiments, the label can also be therapeutic.Various methods of labeling polypeptides and glycoproteins are known inthe art and may be used advantageously in the methods disclosed herein.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,^(99m)Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., fluoresceinisothiocyanate (FITC), rhodamine, or lanthanide phosphors), enzymaticlabels (e.g., horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase), chemiluminescent labels, hapten labels such asbiotinyl groups, and predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). Incertain embodiments, labels are attached by spacer arms (such as(CH₂)_(n), where n<about 20) of various lengths to reduce potentialsteric hindrance.

The term “biological sample” includes, but is not limited to, anyquantity of a substance from a living thing or formerly living thing.Such living things include, but are not limited to, humans, mice,monkeys, rats, rabbits, and other animals. Such substances include, butare not limited to, blood, serum, urine, cells, organs, tissues, bone,bone marrow, lymph, lymph nodes, synovial tissue, chondrocytes, synovialmacrophages, endothelial cells, vascular tissue (particularly inflamedvascular tissue), and skin. The terms “pharmaceutical agent” and “drug”refer to a chemical compound or composition capable of inducing adesired therapeutic effect when properly administered to a patient.

The term “patient” includes human and animal subjects.

Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Amino Acids

The twenty naturally-occurring amino acids and their abbreviationsfollow conventional usage. See Immunology—A Synthesis, 2nd Edition, (E.S. Golub and D. R. Gren, eds.), Sinauer Associates: Sunderland, Mass.(1991), incorporated herein by reference for any purpose. Stereoisomers(e.g., D-amino acids) of the twenty conventional amino acids, unnaturalamino acids such as α-, α-disubstituted amino acids, N-alkyl aminoacids, and other unconventional amino acids may also be suitablecomponents for polypeptides of the invention. Examples of unconventionalamino acids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxyl-terminal direction, in accordance withstandard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA transcriptthat are 5′ to the 5′ end of the RNA transcript are referred to as“upstream sequences”; sequence regions on the DNA strand having the samesequence as the RNA transcript that are 3′ to the 3′ end of the RNAtranscript are referred to as “downstream sequences”.

Naturally occurring amino acid residues may be divided into classesbased on common side chain properties:

-   -   1) hydrophobic: norleucine (Nor or Nle), Met, Ala, Val, Leu,        Ile;    -   2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   3) acidic: Asp, Glu;    -   4) basic: His, Lys, Arg;    -   5) residues that influence chain orientation: Gly, Pro; and    -   6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions may involve exchange of a memberof one of these classes with another member of the same class.Conservative amino acid substitutions may encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of these classes for a member from another class. Such substitutedresidues may be introduced, for example, into regions of a humanantibody that are homologous with non-human antibodies, or into thenon-homologous regions of the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those that are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One may also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Preferred Original Residues ExemplarySubtitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe,Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala,Norlecine

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In other embodiments,the skilled artisan can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In further embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, the skilledartisan can predict the importance of amino acid residues in a proteinthat correspond to amino acid residues important for activity orstructure in similar proteins. One skilled in the art may opt forchemically similar amino acid substitutions for such predicted importantamino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three-dimensional structure. In certain embodiments, one skilledin the art may choose to not make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues maybe involved in important interactions with other molecules. Moreover,one skilled in the art may generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants could be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change can be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof 25 secondary structure. See Moult, 1996, Curr. Op. in Biotech.7:422-427; Chou et al., 1974, Biochemistry 13:222-245; Chou et al.,1974, Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol. Relat.Areas Mol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem.47:251-276; and Chou et al., 1979, Biophys. J. 26:367-384. Moreover,computer programs are currently available to assist with predictingsecondary structure. One method of predicting secondary structure isbased upon homology modeling. For example, two polypeptides or proteinsthat have a sequence identity of greater than 30%, or similarity greaterthan 40% often have similar structural topologies. The recent growth ofthe protein structural database (PDB) has provided enhancedpredictability of secondary structure, including the potential number offolds within a polypeptide's or protein's structure. See Holm et al.,1999, Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner etal., 1997, Curr. Op. Struct. Biol. 7:369-376) that there are a limitednumber of folds in a given polypeptide or protein and that once acritical number of structures have been resolved, structural predictionwill become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See Holm, 1999, supra; and Brenner, 1997, supra).

In certain embodiments, antibody variants include glycosylation variantswherein the number and/or type of glycosylation site has been alteredcompared to the amino acid sequences of the parent polypeptide. Incertain embodiments, protein variants comprise a greater or a lessernumber of N-linked glycosylation sites than the native protein. AnN-linked glycosylation site is characterized by the sequence: Asn-X-Seror Asn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. Additional preferred antibodyvariants include cysteine variants wherein one or more cysteine residuesare deleted from or substituted for another amino acid (e.g., serine)compared to the parent amino acid sequence. Cysteine variants may beuseful when antibodies must be refolded into a biologically activeconformation such as after the isolation of insoluble inclusion bodies.Cysteine variants generally have fewer cysteine residues than the nativeprotein, and typically have an even number to minimize interactionsresulting from unpaired cysteines.

According to certain embodiments, amino acid substitutions are thosethat: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (5) confer ormodify other physicochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts). In preferred embodiments, aconservative amino acid substitution typically does not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles, (Creighton, ed.), 1984, W. H.Freeman and Company, New York; Introduction to Protein Structure (C.Branden and J. Tooze, eds.), 1991, Garland Publishing, New York, N.Y.;and Thornton et al. (1991), Nature 354:105, each of which areincorporated herein by reference.

Preparation of Antibodies

Naturally occurring antibody structural units typically comprise atetramer. Each such tetramer typically is composed of two identicalpairs of polypeptide chains, each pair having one full-length “light”chain (typically having a molecular weight of about 25 kDa) and onefull-length “heavy” chain (typically having a molecular weight of about50-70 kDa). The amino-terminal portion of each chain typically includesa variable region of about 100 to 110 or more amino acids that typicallyis responsible for antigen recognition. The carboxy-terminal portion ofeach chain typically defines a constant region responsible for effectorfunction. Human light chains are typically classified as kappa andlambda light chains. Heavy chains are typically classified as mu, delta,gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. IgG has several subclasses, including,but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclassesincluding, but not limited to, IgM1 and IgM2. IgA is similarlysubdivided into subclasses including, but not limited to, IgA1 and IgA2.Within full-length light and heavy chains, typically, a “J” region ofabout 12 or more amino acids joins the variable region and constantregions, with the heavy chain also including a “D” region of about 10more amino acids. See, e.g., Fundamental Immunology, Ch. 7, 2^(nd) ed.,(Paul, W., ed.), 1989, Raven Press, N.Y. (incorporated by reference inits entirety for all purposes). The combination of the variable regionsof each light chain/heavy chain pair typically forms the antigen-bindingsite.

The variable regions of each of the heavy chains and light chainstypically exhibit the same general structure comprising four relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair typically are aligned by the frameworkregions, which alignment may enable binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chain variable regionstypically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.The assignment of amino acids to each domain is typically in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest (1987 and 1991, National Institutes of Health, Bethesda, Md.),Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917, or Chothia et al.,1989, Nature 342:878-883).

Antibodies became useful and of interest as pharmaceutical agents withthe development of monoclonal antibodies. Monoclonal antibodies areproduced using any method that produces antibody molecules by continuouscell lines in culture. Examples of suitable methods for preparingmonoclonal antibodies include the hybridoma methods of Kohler et al.(1975, Nature 256:495-497) and the human B-cell hybridoma method(Kozbor, 1984, J. Immunol. 13:3001; and Brodeur et al., 1987, MonoclonalAntibody Production Techniques and Applications, (Marcel Dekker, Inc.,New York), pp. 51-63).

Monoclonal antibodies may be modified for use as therapeutics. Oneexample is a “chimeric” antibody in which a portion of the heavy chainand/or light chain is identical with or homologous to a correspondingsequence in antibodies derived from a particular species or belonging toa particular antibody class or subclass, while the remainder of thechain(s) is/are identical with or homologous to a corresponding sequencein antibodies derived from another species or belonging to anotherantibody class or subclass. Other examples are fragments of suchantibodies, so long as they exhibit the desired biological activity.See, U.S. Pat. No. 4,816,567; and Morrison et al. (1985), Proc. Natl.Acad. Sci. USA 81:6851-6855. A related development is the “CDR-grafted”antibody, in which the antibody comprises one or more complementaritydetermining regions (CDRs) from a particular species or belonging to aparticular antibody class or subclass, while the remainder of theantibody chain(s) is/are identical with or homologous to a correspondingsequence in antibodies derived from another species or belonging toanother antibody class or subclass.

Another development is the “humanized” antibody. Methods for humanizingnon-human antibodies are well known in the art. (See U.S. Pat. Nos.5,585,089, and 5,693,762). Generally, a humanized antibody is producedby a non-human animal, and then certain amino acid residues, typicallyfrom non-antigen recognizing portions of the antibody, are modified tobe homologous to said residues in a human antibody of correspondingisotype. Humanization can be performed, for example, using methodsdescribed in the art (Jones et al., 1986, Nature 321:522-525; Riechmannet al., 1988, Nature 332:323-327; Verhoeyen et al., 1988. Science239:1534-1536), by substituting at least a portion of a rodent variableregion for the corresponding regions of a human antibody.

More recent and more promising is the development of human antibodieswithout exposure of antigen to human beings (“fully human antibodies”).Using transgenic animals (e.g., mice) that are capable of producing arepertoire of human antibodies in the absence of endogenous mouseimmunoglobulin production, such antibodies are produced by immunizationwith an antigen (typically having at least 6 contiguous amino acids),optionally conjugated to a carrier. See, for example, Jakobovits et al.,1993, Proc. Natl. Acad. Sci. USA 90:2551-2555; Jakobovits et al., 1993,Nature 362:255-258; and Bruggermann et al., 1993, Year in Immunol. 7:33.In one example of these methods, transgenic animals are produced byincapacitating the endogenous mouse immunoglobulin loci encoding themouse heavy and light immunoglobulin chains therein, and inserting lociencoding human heavy and light chain proteins into the genome thereof.Partially modified animals, which have less than the full complement ofmodifications, are then cross-bred to obtain an animal having all of thedesired immune system modifications. When administered an immunogen,these transgenic animals produce antibodies that are immunospecific forthese antigens having human (rather than murine) amino acid sequences,including variable regions. See PCT Publication Nos. WO96/33735 andWO94/02602, incorporated by reference. Additional methods are describedin U.S. Pat. No. 5,545,807, PCT Publication Nos. WO91/10741, WO90/04036,and in EP 546073B1 and EP 546073A 1, incorporated by reference. Humanantibodies may also be produced by the expression of recombinant DNA inhost cells or by expression in hybridoma cells as described herein.

Fully human antibodies can also be produced from phage-display libraries(as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; andMarks et al., 1991, J. Mol. Biol. 222:581). These processes mimic immuneselection through the display of antibody repertoires on the surface offilamentous bacteriophage, and subsequent selection of phage by theirbinding to an antigen of choice. One such technique is described in PCTPublication No. WO99/10494, incorporated by reference, which describesthe isolation of high affinity and functional agonistic antibodies forMPL- and msk-receptors using such an approach.

Once the nucleotide sequences encoding such antibodies have beendetermined, chimeric, CDR-grafted, humanized, and fully human antibodiesalso may be produced by recombinant methods. Nucleic acids encoding theantibodies are introduced into host cells and expressed using materialsand procedures generally known in the art.

The invention provides one or a plurality of fully human monoclonalantibodies against human IL-1R1. Preferably, the antibodies bind thethird domain of IL-1R1. In preferred embodiments, the invention providesnucleotide sequences encoding, and amino acid sequences comprising,heavy and light chain immunoglobulin molecules, particularly sequencescorresponding to the variable regions thereof. In preferred embodiments,sequences corresponding to complementarity determining regions (CDR's),specifically from CDR1 through CDR3, are provided. In additionalpreferred embodiments, the invention provides hybridoma cell linesexpressing such immunoglobulin molecules and monoclonal antibodiesproduced therefrom, most preferably purified human monoclonal antibodiesagainst human IL-1R1.

The ability to clone and reconstruct megabase-sized human loci in yeastartificial chromosomes (YACs) and to introduce them into the mousegermline provides an advantageous approach to elucidating the functionalcomponents of very large or crudely mapped loci as well as generatinguseful models of human disease. Furthermore, the utilization of suchtechnology for substitution of mouse loci with their human equivalentsprovides unique insights into expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression.

An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B-cell development. Furthermore, such a strategy providesa source for production of fully human monoclonal antibodies (MAbs),particularly for use as therapeutic agents. Fully human antibodies areexpected to minimize the immunogenic and allergic responses intrinsic tomouse or mouse-derivatized Mabs, and to thereby increase the efficacyand safety of administered antibodies in therapeutic applications. Fullyhuman antibodies can be used in the treatment of chronic and recurringhuman diseases, such as osteoarthritis, rheumatoid arthritis, and otherinflammatory conditions, the treatment thereof requiring repeatedantibody administration.

One skilled in the art can engineer mouse strains deficient in mouseantibody production with large fragments of the human Ig loci so thatsuch mice produce human antibodies in the absence of mouse antibodies.Large human Ig fragments may preserve the large variable gene diversityas well as the proper regulation of antibody production and expression.By exploiting the mouse machinery for antibody diversification andselection and the lack of immunological tolerance to human proteins, thereproduced human antibody repertoire in these mouse strains yields highaffinity antibodies against any antigen of interest, including humanantigens. Using the hybridoma technology, antigen-specific human MAbswith the desired specificity may be produced and selected.

In certain embodiments, the skilled artisan can use constant regionsfrom species other than human along with the human variable region(s) insuch mice to produce chimeric antibodies. The antibodies of theinvention can be produced by immunizing such animals with full-lengthIL-1R1, soluble forms of IL-1R1, or a fragment thereof. See, forexample, International Patent Application, Publication WO 93/12227).

The CDRs of the light and heavy chain variable regions of anti-IL-1R1antibodies of the invention can be grafted to framework regions (FRs)from the same, or another, species. In certain embodiments, the CDRs ofthe light and heavy chain variable regions of anti-IL-1R1 antibody maybe grafted to consensus human FRs. To create consensus human FRs, FRsfrom several human heavy chain or light chain amino acid sequences arealigned to identify a consensus amino acid sequence. The FRs of theanti-IL-1R1 antibody heavy chain or light chain can be replaced with theFRs from a different heavy chain or light chain. Rare amino acids in theFRs of the heavy and light chains of anti-IL-1R1 antibody typically arenot replaced, while the rest of the FR amino acids can be replaced. Rareamino acids are specific amino acids that are in positions in which theyare not usually found in FRs. The grafted variable regions fromanti-IL-1R1 antibodies of the invention can be used with a constantregion that is different from the constant region of anti-IL-1R1antibody. Alternatively, the grafted variable regions are part of asingle chain Fv antibody. CDR grafting is described, e.g., in U.S. Pat.Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, whichare hereby incorporated by reference for any purpose.

In certain embodiments, the invention provides anti-IL1-R1 antibodiesthat comprise a human heavy chain CDR1 region having an amino acidsequence of SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 63; a humanheavy chain CDR2 region having an amino acid sequence of SEQ ID NO: 64,SEQ ID NO: 65, or SEQ ID NO: 66; and/or a human heavy chain CDR3 regionhaving an amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 68, or SEQ IDNO: 69.

In other embodiments, the invention provides anti-IL1-R1 antibodies thatcomprise a human light chain CDR1 region having an amino acid sequenceof SEQ ID NO: 70 or SEQ ID NO: 71; a human heavy chain CDR2 regionhaving an amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73; and/ora human heavy chain CDR3 region having an amino acid sequence of SEQ IDNO: 74 or SEQ ID NO: 75.

Antibodies of the invention are preferably prepared using transgenicmice that have a substantial portion of the human antibody-producinglocus inserted in antibody-producing cells of the mice, and that arefurther engineered to be deficient in producing endogenous, murine,antibodies. Such mice are capable of producing human immunoglobulinmolecules and antibodies and do not produce or produce substantiallyreduced amounts of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving this result are disclosed in thepatents, applications, and references disclosed in the specificationherein. In preferred embodiments, the skilled worker may employ methodsas disclosed in International Patent Application Publication No. WO98/24893, which is hereby incorporated by reference for any purpose. Seealso Mendez et al., 1997, Nature Genetics 15:146-156, which is herebyincorporated by reference for any purpose.

The monoclonal antibodies (MAbs) of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein, 1975, Nature 256:495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibodies can be employed, e.g., viral oroncogenic transformation of B-lymphocytes.

In a preferred embodiment, human monoclonal antibodies directed againstIL-1R1 can be generated using mice referred to as “HuMab” mice, containa human immunoglobulin gene minilocus that encodes unrearranged humanheavy (μ and γ) and κ light chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous μ and κ chainloci. Lonberg et al., 1994, Nature 368:856-859. Accordingly, the miceexhibit reduced expression of mouse IgM or κ and in response toimmunization, the introduced human heavy and light chain transgenesundergo class switching and somatic mutation to generate high affinityhuman IgG κ monoclonal antibodies. Lonberg et al., supra; Lonberg andHuszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding and Lonberg, 1995.Ann. N.Y. Acad. Sci. 764:536-546. The preparation of HuMab mice isdescribed in detail in Taylor et al., 1992, Nucleic Acids Res.20:6287-6295; Chen et al., 1993, International Immunology 1:647-656;Tuaillon et al., 1994, J. Immunol. 152:2912-2920; Lonberg et al., 1994,Nature 368:856-859; Lonberg, 1994, Handbook of Exp. Pharmacology113:49-101; Taylor et al., 1994, International Immunology 6:579-591;Lonberg & Huszar, 1995, Intern. Rev. Immunol. 13:65-93; Harding &Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546; Fishwild et al., 1996,Nature Biotechnology 14:845-851, the contents of all of which are herebyincorporated by reference in their entirety. See further U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay,as well as U.S. Pat. No. 5,545,807 to Surani et al.; InternationalPatent Application Publication Nos. WO 93/1227, published Jun. 24, 1993;WO 92/22646, published Dec. 23, 1992; and WO 92/03918, published Mar.19, 1992, the disclosures of all of which are hereby incorporated byreference in their entirety. Alternatively, the HCo7 and HCo12transgenic mice strains described in the Examples below can be used togenerate human anti-IL-1R1 antibodies.

Advantageously, fully human monoclonal antibodies specific for IL-1R1are produced as follows. Transgenic mice containing human immunoglobulingenes are immunized with the IL-1R1-related antigen of interest.Lymphatic cells (such as B-cells) from the mice that express antibodiesare obtained. Such recovered cells are fused with a myeloid-type cellline to prepare immortal hybridoma cell lines, and such hybridoma celllines are screened and selected to identify hybridoma cell lines thatproduce antibodies specific to the antigen of interest. In certainembodiments, the production of a hybridoma cell line that producesantibodies specific to IL-1R1 is provided.

In preferred embodiments, antibodies of the invention are produced byhybridoma lines. In these embodiments, the antibodies of the inventionbind to IL-1R1 with a dissociation constant (K_(d)) of betweenapproximately 4 pM and 100 pM. In certain embodiments of the invention,the antibodies bind to IL-1R1 with a K_(d) of less than about 20 pM. Inother embodiments, the antibodies of the invention bind to the thirddomain of IL-1R1. The nucleotide and amino acid sequences of the thirddomain of human and rat IL-1R1 are shown in FIG. 17.

In preferred embodiments, the antibodies of the invention are of theIgG1, IgG2, or IgG4 isotype, with the IgG2 isotype most preferred. Inpreferred embodiments of the invention, the antibodies comprise a humankappa light chain and a human IgG1, IgG2, or IgG4 heavy chain. Inparticular embodiments, the variable regions of the antibodies areligated to a constant region other than the constant region for theIgG1, IgG2, or IgG4 isotype. In certain embodiments, the antibodies ofthe invention have been cloned for expression in mammalian cells.

In certain embodiments, conservative amino acid substitutions to theheavy and light chains of anti-IL-1R1 antibody (and correspondingmodifications to the encoding nucleotides) will produce anti-IL-1R1antibodies having functional and chemical characteristics similar tothose of anti-IL-1R1 antibody. In contrast, substantial modifications inthe functional and/or chemical characteristics of anti-IL-1R1 antibodymay be accomplished by selecting substitutions in the amino acidsequence of the heavy and light chains that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

For example, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide may also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis” (Wells, 1991,Methods Enzymol. 202:390 (ed. J.J. Langone), Academic Press, London).

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. In certain embodiments, amino acidsubstitutions can be used to identify important residues of anti-IL-1R1antibody, or to increase or decrease the affinity of the anti-IL-1R1antibodies described herein.

In alternative embodiments, antibodies of the invention can be expressedin cell lines other than hybridoma cell lines. In these embodiments,sequences encoding particular antibodies can be used for transformationof a suitable mammalian host cell. According to these embodiments,transformation can be achieved using any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art. Such procedures are exemplified by U.S. Pat. Nos. 4,399,216,4,912,040, 4,740,461, and 4,959,455 (all of which are herebyincorporated herein by reference for any purpose). Generally, thetransformation procedure used may depend upon the host to betransformed. Methods for introducing heterologous polynucleotides intomammalian cells are well known in the art and include, but are notlimited to, dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

According to certain embodiments of the methods of the invention, anucleic acid molecule encoding the amino acid sequence of a heavy chainconstant region, a heavy chain variable region, a light chain constantregion, or a light chain variable region of an IL-1R1 antibody of theinvention is inserted into an appropriate expression vector usingstandard ligation techniques. In a preferred embodiment, the IL-1R1heavy or light chain constant region is appended to the C-terminus ofthe appropriate variable region and is ligated into an expressionvector. The vector is typically selected to be functional in theparticular host cell employed (i.e., the vector is compatible with thehost cell machinery such that amplification of the gene and/orexpression of the gene can occur). For a review of expression vectors,see, Goeddel (ed.), 1990, Meth. Enzymol. Vol. 185, Academic Press. N.Y.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the IL-1R1polypeptide coding sequence; the oligonucleotide sequence encodespolyHis (such as hexaHis (SEQ ID NO: 90), or another “tag” such as FLAG,HA (hemaglutinin influenza virusO, or myc for which commerciallyavailable antibodies exist. This tag is typically fused to thepolypeptide upon expression of the polypeptide, and can serve as a meansfor affinity purification or detection of the IL-1R1 antibody from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified IL-1R1 polypeptide by various means such as using certainpeptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria andvarious viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in both prokaryotic and eukaryotic host cells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are ingreater demand for the production of a protein critical for growth orcell survival are reiterated generally in tandem within the chromosomesof successive generations of recombinant cells. Examples of suitableselectable markers for mammalian cells include dihydrofolate reductase(DHFR) and promoterless thymidine kinase. Mammalian cell transformantsare placed under selection pressure wherein only the transformants areuniquely adapted to survive by virtue of the selectable gene present inthe vector. Selection pressure is imposed by culturing the transformedcells under conditions in which the concentration of selection agent inthe medium is successively increased, thereby leading to theamplification of both the selectable gene and the DNA that encodesanother gene, such as IL-1R1 polypeptide comprising the vector. As aresult, increased quantities of a polypeptide such as IL-1R1 polypeptideare synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addpro-sequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

The expression and cloning vectors of the invention will typicallycontain a promoter that is recognized by the host organism and operablylinked to the molecule encoding the anti-IL-1R1 antibody. Promoters areuntranscribed sequences located upstream (i.e., 5′) to the start codonof a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,initiate continual gene product production; that is, there is little orno control over gene expression. A large number of promoters, recognizedby a variety of potential host cells, are well known. A suitablepromoter is operably linked to the DNA encoding heavy chain or lightchain comprising an anti-IL-1R1 antibody of the invention by removingthe promoter from the source DNA by restriction enzyme digestion andinserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290:304-10); the CMV promoter, the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell22:787-97); the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. USA 78:1444-45); the regulatory sequences of themetallothionine gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-31); orthe tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA80:21-25). Also of interest are the following animal transcriptionalcontrol regions, which exhibit tissue specificity and have been utilizedin transgenic animals: the elastase I gene control region that is activein pancreatic acinar cells (Swift et al., 1984, Cell 38:639-46; Ornitzet al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986);MacDonald, 1987, Hepatology 7:425-515); the insulin gene control regionthat is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-22); the immunoglobulin gene control region that is active inlymphoid cells (Grosschedl et al., 1984. Cell 38:647-58; Adames et al.,1985, Nature 318:533-38; Alexander et al., 1987, Mol. Cell. Biol.7:1436-44); the mouse mammary tumor virus control region that is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-95); the albumin gene control region that is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-76); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-48; Hammer et al., 1987, Science235:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-71); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-12); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-86); and the gonadotropicreleasing hormone gene control region that is active in the hypothalamus(Mason et al., 1986, Science 234:1372-78).

An enhancer sequence may be inserted into the vector to increasetranscription of DNA encoding light chain or heavy chain comprising ananti-IL-1R1 antibody of the invention by higher eukaryotes. Enhancersare cis-acting elements of DNA, usually about 10-300 bp in length, thatact on the promoter to increase transcription. Enhancers are relativelyorientation- and position-independent. They have been found 5′ and 3′ tothe transcription unit. Several enhancer sequences available frommammalian genes (e.g., globin, elastase, albumin, alpha-feto-protein andinsulin) are known. Typically, however, an enhancer from a virus isused. The SV40 enhancer, the cytomegalovirus early promoter enhancer,the polyoma enhancer, and adenovirus enhancers known in the art areexemplary enhancing elements for the activation of eukaryotic promoters.While an enhancer may be spliced into the vector at a position 5′ or 3′to a nucleic acid molecule, it is typically located at a site 5′ fromthe promoter.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding light chain or heavy chain or light chain and heavy chaincomprising an anti-IL-1R1 antibody has been inserted into the propersite of the vector, the completed vector may be inserted into a suitablehost cell for amplification and/or polypeptide expression. Thetransformation of an expression vector for an anti-IL-1R1 antibody intoa selected host cell may be accomplished by well known methods includingtransfection, infection, calcium phosphate co-precipitation,electroporation, microinjection, lipofection, DEAE-dextran mediatedtransfection, or other known techniques. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., supra.

The host cell, when cultured under appropriate conditions, synthesizesan anti-IL-1R1 antibody that can subsequently be collected from theculture medium (if the host cell secretes it into the medium) ordirectly from the host cell producing it (if it is not secreted). Theselection of an appropriate host cell will depend upon various factors,such as desired expression levels, polypeptide modifications that aredesirable or necessary for activity (such as glycosylation orphosphorylation) and ease of folding into a biologically activemolecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, many immortalized celllines available from the American Type Culture Collection (A.T.C.C.),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, one may select cell lines bydetermining which cell lines have high expression levels and produceantibodies with constitutive IL-1R1 binding properties. In anotherembodiment, one may select a cell line from the B cell lineage that doesnot make its own antibody but has a capacity to make and secrete aheterologous antibody (e.g., mouse myeloma cell lines NS0 and SP2/0).

Antibodies of the invention are useful for detecting IL-1R1 inbiological samples and identification of cells or tissues that produceIL-1R1 protein. Said antibodies that bind to IL-1R1 and blockinteraction with other binding compounds have therapeutic use inmodulating IL-1 mediated diseases. In preferred embodiments, antibodiesto IL-1R1 can block IL-1R1 binding to IL-1β or IL-1α, which can resultin disruption of the IL-1 signal transduction cascade.

Antibodies of the invention that specifically bind to IL-1R1 may beuseful in treatment of IL-1 mediated diseases, as discussed below. Saidantibodies can be used in binding assays to detect IL-1R1 binding andtheir capacity to inhibit IL-1R1 from forming a complex with IL-1β andIL-1R accessory protein (IL-1RAcP) or with IL-1α and IL-1RacP.

In certain embodiments, the invention provides methods for treatingmedical disorders associated with IL-1 mediated inflammatory reactionsor IL-1 mediated immunoregulatory reactions. The methods of theinvention include administering an anti-IL1R1 antibody of the inventionto an individual who is afflicted with an inflammatory orimmunoregulatory disease that is mediated by IL-1. As used herein, theterms “illness,” “disease,” “medical condition,” or “abnormalcondition,” are used interchangeably with the term “medical disorder.”

In a particular embodiment, the methods of the invention involveadministering to a patient an anti-IL-1R1 antibody of the invention,thereby preventing the binding of IL-1 to its cell surface receptor(IL-1R1).

To treat a medical disorder characterized by abnormal or excessexpression of IL-1 or abnormal or excess IL-1 signaling, a moleculecomprising an IL-1R type I antibody of this invention is administered tothe patient in an amount and for a time sufficient to induce a sustainedimprovement in at least one indicator that reflects the severity of thedisorder. An improvement is considered “sustained” if the patientexhibits the improvement on at least two occasions separated by one tofour weeks. The degree of improvement is determined based on signs orsymptoms, and may also employ questionnaires that are administered tothe patient, such as quality-of-life questionnaires.

Various indicators that reflect the extent of the patient's illness maybe assessed for determining whether the amount and time of the treatmentis sufficient. The baseline value for the chosen indicator or indicatorsis established by examination of the patient prior to administration ofthe first dose of the antibody. Preferably, the baseline examination isdone within about 60 days of administering the first dose. If the IL-1Rantibody is being administered to treat acute symptoms, such as, forexample, to treat traumatic injuries (traumatic knee injury, stroke,head injury, etc.) the first dose is administered as soon as practicallypossible after the injury or event has occurred.

Improvement is induced by repeatedly administering a dose of antibodyuntil the patient manifests an improvement over baseline for the chosenindicator or indicators. In treating chronic conditions, this degree ofimprovement is obtained by repeatedly administering this medicament overa period of at least a month or more, e.g., for one, two, or threemonths or longer, or indefinitely. A period of one to six weeks, or evena single dose, often is sufficient for treating acute conditions.

Although the extent of the patient's illness after treatment may appearimproved according to one or more indicators, treatment may be continuedindefinitely at the same level or at a reduced dose or frequency. Oncetreatment has been reduced or discontinued, it later may be resumed atthe original level if symptoms should reappear.

Any efficacious route of administration may be used to therapeuticallyadminister the antibody. The antibody may be injected viaintra-articular, intravenous, intramuscular, intralesional,intraperitoneal, intracranial, inhalation or subcutaneous routes bybolus injection or by continuous infusion. For example, pulmonarydiseases can involve intranasal and inhalation methods. Other suitablemeans of administration include sustained release from implants, aerosolinhalation, eyedrops, oral preparations, including pills, syrups,lozenges or chewing gum, and topical preparations such as lotions, gels,sprays, ointments or other suitable techniques. Administration byinhalation is particularly beneficial when treating diseases associatedwith pulmonary disorders.

In one embodiment of the invention, an anti-IL-1R1 antibody of theinvention can be administered once a month. In another embodiment theantibody is administered once every two weeks or one time per week totreat the various medical disorders disclosed herein. In yet anotherembodiment the antibody is administered at least two times per week, andin another embodiment is administered at least once per day. An adultpatient is a person who is 18 years of age or older. If injected, theeffective amount, per adult dose, ranges from 1-200 mg/m², or from 1-40mg/m² or about 5-25 mg/m². Alternatively, a flat dose may beadministered, whose amount may range from 2-400 mg/dose, 2-100 mg/doseor from about 10-80 mg/dose. If the dose is to be administered more thanone time per week, an exemplary dose range is the same as the foregoingdescribed dose ranges or lower. In one embodiment of the invention, thevarious indications described below are treated by administering apreparation acceptable for injection containing IL-1 receptor antibodyat 80-100 mg/dose, or alternatively, containing 80 mg per dose. The doseis administered repeatedly. If a route of administration other thaninjection is used, the dose is appropriately adjusted in accord withstandard medical practices. For example, if the route of administrationis inhalation, dosing may be one to seven times per week at dose rangesfrom 10 mg/dose to 50 mg per dose.

In preferred embodiments, the invention also provides pharmaceuticalcompositions comprising a therapeutically effective amount of one or aplurality of the antibodies of the invention together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant. Preferably, acceptable formulationmaterials are nontoxic to recipients at the dosages and concentrationsemployed. In preferred embodiments, pharmaceutical compositionscomprising a therapeutically effective amount of anti-IL-1R1 antibodiesare provided.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, trimethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,Remington's Pharmaceutical Sciences, 18^(th) Edition, (A.R. Gennaro,ed.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition may be either aqueous or non-aqueous innature. For example, a suitable vehicle or carrier may be water forinjection, physiological saline solution or artificial cerebrospinalfluid, possibly supplemented with other materials common in compositionsfor parenteral administration. Neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In preferredembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may furtherinclude sorbitol or a suitable substitute therefor. In certainembodiments of the invention, anti-IL-1R1 antibody compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, the anti-IL-1R1antibody product may be formulated as a lyophilizate using appropriateexcipients such as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. The compositions may be selected for inhalation orfor delivery through the digestive tract, such as orally. Preparation ofsuch pharmaceutically acceptable compositions is within the skill of theart.

The formulation components are present preferably in concentrations thatare acceptable to the site of administration. In certain embodiments,buffers are used to maintain the composition at physiological pH or at aslightly lower pH, typically within a pH range of from about 5 to about8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired anti-IL-1R1 antibody in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the anti-IL-1R1 antibody is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe product which can be delivered via depot injection. In certainembodiments, hyaluronic acid may also be used, having the effect ofpromoting sustained duration in the circulation. In certain embodiments,implantable drug delivery devices may be used to introduce the desiredantibody molecule.

Pharmaceutical compositions of the invention can be formulated forinhalation. In these embodiments, anti-IL-1R1 antibodies are formulatedas a dry powder for inhalation. In preferred embodiments, anti-IL-1R1antibody inhalation solutions may also be formulated with a propellantfor aerosol delivery. In certain embodiments, solutions may benebulized. Pulmonary administration and formulation methods thereforeare further described in International Patent Publication No.WO94/20069, incorporated by reference, which describes pulmonarydelivery of chemically modified proteins.

It is also contemplated that formulations can be administered orally.Anti-IL-1R1 antibodies that are administered in this fashion can beformulated with or without carriers customarily used in the compoundingof solid dosage forms such as tablets and capsules. In certainembodiments, a capsule may be designed to release the active portion ofthe formulation at the point in the gastrointestinal tract whenbioavailability is maximized and pre-systemic degradation is minimized.Additional agents can be included to facilitate absorption of theanti-IL-1R1 antibody. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders may also be employed.

A pharmaceutical composition of the invention is preferably provided tocomprise an effective quantity of one or a plurality of anti-IL-1R1antibodies in a mixture with non-toxic excipients that are suitable forthe manufacture of tablets. By dissolving the tablets in sterile water,or another appropriate vehicle, solutions may be prepared in unit-doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving anti-IL-1R1 antibodies insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. Seefor example, International Patent Publication No. WO93/15722,incorporated by reference, which describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g. films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481), copolymers of L-glutamicacid and gamma ethyl-L-glutamate (Sidman et al., 1983, Biopolymers22:547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J.Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (European Patent Application PublicationNo. EP 133,988). Sustained release compositions may also includeliposomes that can be prepared by any of several methods known in theart. See e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA82:3688-3692; European Patent Application Publication Nos. EP 036,676;EP 088,046 and EP 143,949.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations may bestored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration.

The invention also provides kits for producing a single-doseadministration unit. The kits of the invention may each contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments of this invention, kitscontaining single and multi-chambered pre-filled syringes (e.g., liquidsyringes and lyosyringes) are provided.

The effective amount of an anti-IL-1R1 antibody-containingpharmaceutical composition to be employed therapeutically will depend,for example, upon the therapeutic context and objectives. One skilled inthe art will appreciate that the appropriate dosage levels for treatmentwill vary depending, in part, upon the molecule delivered, theindication for which the anti-IL-1R1 antibody is being used, the routeof administration, and the size (body weight, body surface or organsize) and/or condition (the age and general health) of the patient. Incertain embodiments, the clinician may titer the dosage and modify theroute of administration to obtain the optimal therapeutic effect. Atypical dosage may range from about 0.1 μg/kg to up to about 100 mg/kgor more, depending on the factors mentioned above. In preferredembodiments, the dosage may range from 0.1 μg/kg up to about 100 mg/kg;more preferably from 1 μg/kg up to about 100 mg/kg; or even morepreferably from 5 μg/kg up to about 100 mg/kg.

Dosing frequency will depend upon the pharmacokinetic parameters of theparticular anti-IL-1R1 antibody in the formulation used. Typically, aclinician administers the composition until a dosage is reached thatachieves the desired effect. The composition may therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages may be ascertained through use ofappropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g. orally, through injection byintravenous, intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; by sustained release systems or byimplantation devices. In certain embodiments, the compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device.

The composition also may be administered locally via implantation of amembrane, sponge or another appropriate material onto which the desiredmolecule has been absorbed or encapsulated. In certain embodiments,where an implantation device is used, the device may be implanted intoany suitable tissue or organ, and delivery of the desired molecule maybe via diffusion, timed-release bolus, or continuous administration.

It also may be desirable to use anti-IL-1R1 antibody pharmaceuticalcompositions according to the invention ex vivo. In such instances,cells, tissues or organs that have been removed from the patient areexposed to anti-IL-1R1 antibody pharmaceutical compositions after whichthe cells, tissues and/or organs are subsequently implanted back intothe patient.

In particular, anti-IL-1R1 antibodies can be delivered by implantingcertain cells that have been genetically engineered, using methods suchas those described herein, to express and secrete the polypeptide. Incertain embodiments, such cells may be animal or human cells, and may beautologous, heterologous, or xenogeneic. In certain embodiments, thecells may be immortalized. In other embodiments, in order to decreasethe chance of an immunological response, the cells may be encapsulatedto avoid infiltration of surrounding tissues. In further embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

In certain embodiments, the invention further encompasses theadministration of an anti-IL-1R1 antibody or pharmaceutical compositionof the invention concurrently with one or more other drugs that areadministered to the same patient, each drug being administered accordingto a regimen suitable for that medicament. This encompassespre-treatment, simultaneous treatment, sequential treatment andalternating regimens. Examples of such drugs include, but are notlimited to, antivirals, antibiotics, analgesics, corticosteroids,antagonists of inflammatory cytokines, disease-modifying anti-rheumaticdrugs (DMARDs), and non-steroidal anti-inflammatories.

In other embodiments, an anti-IL-1R1 antibody or pharmaceuticalcomposition of the invention can be administered in combination withother cytokine inhibitors, including those that antagonize, for example,RANKL, TGFβ, IFNγ, IL-6 or IL-8 and TNF, particularly TNFα. Incombination with IL-6, an antibody of this invention can be used totreat and prevent the recurrence of seizures, including seizures inducedby GABA_(A) receptor antagonism, seizures associated with EEG ictalepisodes and motor limbic seizures occurring during status epilepticus.In combination with IFNγ inhibitor, an antibody of this invention isuseful in treating idiopathic pulmonary fibrosis and cystic fibrosis.The combination of an IL-1 receptor antibody and RANKL inhibitors, e.g.a RANKL antibody is useful for preventing bone destruction in varioussettings including but not limited to various rheumatic disorders,osteoporosis, multiple myeloma or other malignancies that cause bonedegeneration, or anti-tumor therapy aimed at preventing metastasis tobone, or bone destruction associated with prosthesis wear debris or withperiodontitis. In addition, antibodies of the invention may beadministered in combination with IL-17 inhibitors such soluble forms ofan IL-17 receptor (such as IL-17R:Fc) or an IL-17 antibody or IL-17Rantibody, IL-18 binding protein, soluble forms of IL-18 receptors, andIL-18 antibodies, antibodies against IL-18 receptors or antibodiesagainst CD30-ligand or against CD4.

The invention further encompasses methods for using an anti-IL1R1antibody or pharmaceutical composition of the invention in treating theherein disclosed medical disorders in combination with a TNF inhibitor,preferably TNFR:Fc (ENBREL®) and any combination of the above describedcytokines or cytokine inhibitors that are active agents in combinationtherapies. For example, in accordance with the present invention,combination therapy methods may be used for treating rheumatoidarthritis, stroke, asthma, psoriasis, etc.

Conditions effectively treated by an anti-IL-1R1 antibody orpharmaceutical composition described herein include pulmonary diseasessuch as asthma, chronic obstructive pulmonary disease, pulmonaryalveolar proteinosis, bleomycin-induced pneumopathy and fibrosis,radiation-induced pulmonary fibrosis, cystic fibrosis, collagenaccumulation in the lungs, and ARDS, all of which may be treated withcombinations of an antibody to IL-1R and an IL-4 inhibitor and/or IL-13inhibitor, e.g. IL-4R antibody that inhibits IL-13 and IL-4 activity.The disclosed antibodies and pharmaceutical compositions of theinvention also are useful for treating broncho-pulmonary dysplasia(BPD); chronic obstructive pulmonary diseases (e.g. emphysema andchronic bronchitis), and chronic fibrotic lung disease of preterminfants. In addition, the compounds, compositions and combinationtherapies of the invention are used to treat occupational lung diseases,including asbestosis, coal worker's pneumoconiosis, silicosis or similarconditions associated with long-term exposure to fine particles. Inother aspects of the invention, the disclosed compounds, compositionsand combination therapies are used to treat bronchioliterans organizingpneumonia, pulmonary fibrosis, including idiopathic pulmonary fibrosisand radiation-induced pulmonary fibrosis; pulmonary sarcoidosis; andallergies, including allergic rhinitis, contact dermatitis, atopicdermatitis and asthma.

Such combinations are useful also for treating patients suffering fromvarious skin disorders, including but not limited to dermatitisherpetiformis (Duhring's disease), atopic dermatitis, contactdermatitis, urticaria (including chronic idiopathic urticaria), andautoimmune blistering diseases, including pemphigus vulgaris and bullouspemphigoid. Other diseases treatable with the combination of an IL-1Rantibody and an IL-4 and/or IL-13 inhibitor include myesthenia gravis,sarcoidosis, including pulmonary sarcoidosis, scleroderma, reactivearthritis, hyper IgE syndrome, multiple sclerosis and idiopathichypereosinophil syndrome. The combination is used also for treatingallergic reactions to medication and as an adjuvant to allergyimmunotherapy.

The IL-1 receptor antibodies and pharmaceutical compositions describedherein are useful for treating protozoal diseases, including malaria andschistosomiasis and to treat erythema nodosum leprosum; bacterial orviral meningitis; tuberculosis, including pulmonary tuberculosis; andpneumonitis secondary to a bacterial or viral infection includinginfluenza infection and infectious mononucleosis.

Cardiovascular disorders and injuries are treatable and/or preventablewith disclosed either pharmaceutical compositions or anti-IL1-R1antibodies alone or in combination with other cytokine inhibitors.Cardiovascular disorders treatable include aortic aneurysms; includingabdominal aortic aneurysms, acute coronary syndrome, arteritis; vascularocclusion, including cerebral artery occlusion; complications ofcoronary by-pass surgery; ischemia/reperfusion injury; heart disease,including atherosclerotic heart disease, myocarditis, including chronicautoimmune myocarditis and viral myocarditis; heart failure, includingchronic heart failure, congestive heart failure, cachexia of heartfailure; myocardial infarction; restenosis and/or atherosclerosis afterheart surgery or after carotid artery balloon angioplastic procedures;silent myocardial ischemia; left ventricular pump dysfunction, postimplantation complications of left ventricular assist devices; Raynaud'sphenomena; thrombophlebitis; vasculitis, including Kawasaki'svasculitis; veno-occlusive disease, giant cell arteritis, Wegener'sgranulomatosis; mental confusion following cardio pulmonary by passsurgery, and Schoenlein-Henoch purpura.

In certain embodiments, anti-IL-1R1 antibodies and pharmaceuticalcompositions of the invention can also be used to treat chronic painconditions, such as chronic pelvic pain, including chronicprostatitis/pelvic pain syndrome, and post-herpetic pain.

Disorders of the endocrine system including juvenile onset diabetes(includes autoimmune diabetes mellitus and insulin-dependent types ofdiabetes) and maturity onset diabetes (includes non-insulin dependentand obesity-mediated diabetes) can also be treated with anti-IL-1R1antibodies or pharmaceutical compositions of the invention. Suchtreatment includes secondary conditions associated with diabetes, suchas diabetic retinopathy, kidney transplant rejection in diabeticpatients, obesity-mediated insulin resistance, and renal failure, whichitself may be associated with proteinurea and hypertension. Otherendocrine disorders also are treatable with these compounds and includepolycystic ovarian disease, X-linked adrenoleukodystrophy,hypothyroidism and thyroiditis, including Hashimoto's thyroiditis (i.e.,autoimmune thyroiditis), thyroid cell dysfunction, including euthyroidsick syndrome.

Conditions of the gastrointestinal system are treatable or preventablewith anti-IL-1R1 antibodies or pharmaceutical compositions of theinvention, alone or in combination with other therapeutics. Theseconditions include coeliac disease, Crohn's disease; ulcerative colitis;idiopathic gastroparesis; pancreatitis, including chronic pancreatitis;acute pancreatitis, inflammatory bowel disease and ulcers, includinggastric and duodenal ulcers.

Disorders of the genitourinary system are also treatable or preventablewith the anti-IL-1R1 antibodies or pharmaceutical compositions describedherein. Such disorders include glomerulonephritis, including autoimmuneglomerulonephritis, glomerulonephritis due to exposure to toxins orglomerulonephritis secondary to infections with haemolytic streptococcior other infectious agents. Also treatable with the compounds,compositions and combination therapies of the invention are uremicsyndrome and its clinical complications (for example, renal failure,anemia, and hypertrophic cardiomyopathy), including uremic syndromeassociated with exposure to environmental toxins, drugs or other causes.Complications that arise from inflammation of the gallbladder wall thatleads to alteration in absorptive function are treatable or preventablewith the antibodies of this invention. Included in such complicationsare cholelithiasis (gallstones) and choliedocholithiasis (bile ductstones) and the recurrence of cholelithiasis and choliedocholithiasis.Further conditions treatable with the compounds, compositions andcombination therapies of the invention are complications ofhemodialysis; prostate conditions, including benign prostatichypertrophy, nonbacterial prostatitis and chronic prostatitis; andcomplications of hemodialysis.

Also provided herein are methods for using anti-IL-1R1 antibodies of theinvention, compositions, and combination therapies to treat varioushematologic and oncologic disorders. For example, anti-IL-1R1antibodies, alone or in combination with other cytokine inhibitors orother active agents as described above, can be used to treat variousforms of cancer, including acute myelogenous leukemia, chronicmyelogenous leukemia, Epstein-Barr virus-positive nasopharyngealcarcinoma, glioma, colon, stomach, prostate, renal cell, cervical andovarian cancers, lung cancer (SCLC and NSCLC), includingcancer-associated cachexia, fatigue, asthenia, paraneoplastic syndromeof cachexia and hypercalcemia. Solid tumors, including sarcoma,osteosarcoma, and carcinoma, such as adenocarcinoma (for example, breastcancer) and squamous cell carcinoma are also treatable. Additionaltreatable cancers include esophogeal cancer, gastric cancer, gallbladder carcinoma, leukemia, including acute myelogenous leukemia,chronic myelogenous leukemia, myeloid leukemia, chronic or acutelymphoblastic leukemia and hairy cell leukemia. Other malignancies withinvasive metastatic potential, including multiple myeloma, can betreated with the subject compounds, compositions and combinationtherapies.

In addition, the disclosed anti-IL-1R1 antibodies can be used to treatanemias and hematologic disorders, including chronic idiopathicneutropenia, anemia of chronic disease, aplastic anemia, includingFanconi's aplastic anemia; idiopathic thrombocytopenic purpura (ITP);thrombotic thrombocytopenic purpura, myelodysplastic syndromes(including refractory anemia, refractory anemia with ringedsideroblasts, refractory anemia with excess blasts, refractory anemiawith excess blasts in transformation); myelofibrosis/myeloid metaplasia;and sickle cell vasocclusive crisis.

Various lymphoproliferative disorders also are treatable withanti-IL-1R1 antibodies of the invention, including autoimmunelymphoproliferative syndrome (ALPS), chronic lymphoblastic leukemia,hairy cell leukemia, chronic lymphatic leukemia, peripheral T-celllymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicularlymphoma, Burkitt's lymphoma, Epstein-Barr virus-positive T celllymphoma, histiocytic lymphoma. Hodgkin's disease, diffuse aggressivelymphoma, acute lymphatic leukemias, T gamma lymphoproliferativedisease, cutaneous B cell lymphoma, cutaneous T cell lymphoma (i.e.,mycosis fungoides) and Sézary syndrome.

Hereditary conditions such as Gaucher's disease, Huntington's disease,linear IgA disease, and muscular dystrophy are treatable with theantibodies of this invention.

Other conditions treatable or preventable by the disclosed IL-1 receptorantibodies or pharmaceutical compositions include those resulting frominjuries to the head or spinal cord including subdural hematoma due totrauma to the head. In connection with this therapy, the compositionsand combinations described are suitable for preventing cranialneurologic damage and preventing and treating cervicogenic headache. Thecompositions and combinations described are further suitable fortreating neurological side effects associated with brain irradiation.

Anti-IL-1R1 antibodies and pharmaceutical composition of the inventionare also useful for treating conditions of the liver such as hepatitis,including acute alcoholic hepatitis, acute drug-induced or viralhepatitis, hepatitis A, B and C, sclerosing cholangitis, hepaticsinusoid epithelium, and inflammation of the liver due to unknowncauses.

Disorders that involve hearing loss and that are associated withabnormal IL-1 expression are treatable with the anti-IL-1R1 antibodiesor pharmaceutical compositions of the invention. Such disorders includecochlear nerve-associated hearing loss that is thought to result from anautoimmune process, i.e., autoimmune hearing loss. Also treatable orpreventable with the anti-IL-1R1 antibodies or pharmaceuticalcompositions of the invention is Meniere's syndrome and cholesteatoma, amiddle ear disorder often associated with hearing loss.

Non-arthritic disorders of the bones and joints and also treatable withthe antibodies described herein. This encompasses osteoclast disordersthat lead to bone loss, such as but not limited to osteoporosis,including post-menopausal osteoporosis, osteoarthritis, periodontitisresulting in tooth loosening or loss, and prosthesis loosening afterjoint replacement (generally associated with an inflammatory response towear debris). This latter condition also is called “orthopedic implantosteolysis.” Another condition treatable with the compounds,compositions and combination therapies of the invention is temporalmandibular joint dysfunction (TMJ).

The anti-IL-1R1 antibodies or pharmaceutical compositions of theinvention can also be used to treat rheumatic disorders including adultand juvenile rheumatoid arthritis; scleroderma; systemic lupuserythematosus; gout; osteoarthritis; polymyalgia rheumatica;seronegative spondylarthropathies, including ankylosing spondylitis, andReiter's disease, psoriatic arthritis and chronic Lyme arthritis. Theantibodies of this invention are also useful for treating inflammationof the voluntary muscle and other muscles, including dermatomyositis,inclusion body myositis, polymyositis, and lymphangioleimyomatosis.

Another use for the antibodies and pharmaceutical compositions of theinvention is the treatment and/or prevention of primary amyloidosis andthe secondary amyloidosis that is characteristic of various conditionincluding Alzheimer's disease, secondary reactive amyloidosis; Down'ssyndrome; and dialysis-associated amyloidosis. Also treatable with theantibodies or pharmaceutical compositions of the invention are inheritedperiodic fever syndromes, including familial Mediterranean fever,hyperimmunoglobulin D and periodic fever syndrome and TNF-receptorassociated periodic syndromes (TRAPS).

In other embodiments, the antibodies or pharmaceutical compositions ofthe invention can be used to treat disorders involving the skin ormucous membranes. Such disorders include acantholytic diseases,including Darier's disease, keratosis follicularis and pemphigusvulgaris. Additional skin disorders that can be treated using antibodiesof the invention include acne, acne rosacea, alopecia areata, aphthousstomatitis, bullous pemphigoid, burns, eczema, erythema, includingerythema multiforme and erythema multiforme bullosum (Stevens-Johnsonsyndrome), inflammatory skin disease, lichen planus, linear IgA bullousdisease (chronic bullous dermatosis of childhood), loss of skinelasticity, mucosal surface ulcers, including gastric ulcers,neutrophilic dermatitis (Sweet's syndrome), dermatomyositis, pityriasisrubra pilaris, psoriasis, pyoderma gangrenosum, multicentricreticulohistiocytosis, and toxic epidermal necrolysis. Other skinrelated conditions treatable by the therapies and combination therapiesof the present invention include dermatitis herpetiformis.

Additional disorders that can be treated with the antibodies orpharmaceutical compositions of the invention include graft-versus-hostdisease, and complications resulting from solid organ transplantation,such as heart, liver, skin, kidney, lung (lung transplant airwayobliteration) or other transplants, including bone marrow transplants.

Ocular disorders also are treatable or preventable with the disclosedanti-IL-1R1 antibodies or pharmaceutical compositions, includingrhegmatogenous retinal detachment, and inflammatory eye disease,including inflammatory eye disease associated with smoking and maculardegeneration.

Antibodies or pharmaceutical compositions of the invention, as describedherein, are useful for treating disorders that affect the femalereproductive system. Examples include, but are not limited to, multipleimplant failure/infertility; fetal loss syndrome or IV embryo loss(spontaneous abortion); preeclamptic pregnancies or eclampsia;endometriosis, chronic cervicitis, and pre-term labor.

In addition, the antibodies or pharmaceutical compositions of theinvention are useful for treating and/or preventing sciatica, symptomsof aging, severe drug reactions (for example, Il-2 toxicity orbleomycin-induced pneumopathy and fibrosis), or to suppress theinflammatory response prior, during or after the transfusion ofallogeneic red blood cells in cardiac or other surgery, or in treating atraumatic injury to a limb or joint, such as traumatic knee injury.Various other medical disorders treatable with the disclosed anti-IL-1R1antibodies or pharmaceutical compositions include; multiple sclerosis;Behcet's syndrome; Sjogren's syndrome; autoimmune hemolytic anemia; betathalassemia; amyotrophic lateral sclerosis (Lou Gehrig's Disease);Parkinson's disease; and tenosynovitis of unknown cause, as well asvarious autoimmune disorders or diseases associated with hereditarydeficiencies, including x-linked mental retardation.

Furthermore, the anti-IL-1R1 antibodies or pharmaceutical compositionsof the invention are useful for treating central nervous system (CNS)injuries, including the effects of neurotoxic neurotransmittersdischarged during excitation of inflammation in the central nervoussystem and to inhibit or prevent the development of glial scars at sitesof central nervous system injury. In connection with epilepsy and thetreatment of seizures, reducing the severity and number of recurringseizures, and reducing the severity of the deleterious effects ofseizures, reducing neuronal loss, neuronal degeneration, and gliosisassociated with seizures.

Additional uses for the antibodies or pharmaceutical compositions of theinvention include, but are limited to, treating critical illnesspolyneuropathy and myopathy (CIPNM) acute polyneuropathy; anorexianervosa; Bell's palsy; chronic fatigue syndrome; transmissible dementia,including Creutzfeld-Jacob disease; demyelinating neuropathy;Guillain-Barre syndrome; vertebral disc disease; Gulf war syndrome;chronic inflammatory demyelinating polyneuropathy, myasthenia gravis;silent cerebral ischemia; sleep disorders, including narcolepsy andsleep apnea; chronic neuronal degeneration; and stroke, includingcerebral ischemic diseases. Still additional uses for the antibodies ofthe invention are anorexia and/or anorexic conditions, peritonitis,endotoxemia and septic shock, granuloma formation, heat stroke,Churg-Strauss syndrome, chronic inflammation following acute infectionssuch as tuberculosis and leprosy, systemic sclerosis and hypertrophicscarring.

In other embodiments, avidin fusion proteins comprising an amino acidsequence of one of the IL-1R1 antibodies of the invention can beconstructed for various purposes. Avidin fusion proteins can begenerated, for example, using a mammalian expression vector containingcDNA sequence encoding recombinant chicken avidin adjacent to a multiplecloning site for insertion of a specific target gene fusion partner. Thevector can include an avidin sequence with its endogenous signalsequence to enable secretion of discrete fusion gene partners that donot naturally contain signal sequences. The fusion protein expressed bythe vector has an avidin protein tag at the N-terminal portion of thefusion partner. The fusion strategy as described herein has thecapability of secreting proteins that are normally expressedintracellularly, such as signal transduction genes or nuclear hormonereceptors.

Alternatively, a vector can be used that encodes avidin without itsendogenous signal sequence, which will result in C-terminal tagging offusion protein partners. A C-terminal avidin fusion also allows forprotein secretion based on the endogenous signal sequence of the fusionpartner. Such a strategy can be applied to allow for correct proteinprocessing and folding or to determine validity of a proposed signalsequence. Additionally, the vector can comprise a short nucleotidesequence encoding an amino acid sequence, which can act as a specificenzyme-cleavable substrate, between the avidin and fusion partnersequences. Such enzyme-cleavable sequences allow for separation of thefusion partner from the avidin for purification or protein releasepurposes.

Avidin fusion proteins of the invention can be used, for example, inantibody screening, functional characterization (determination of anantibody's utility as an agonist or antagonist, neutralizing agent,etc.), epitope mapping, or immunization strategies. Avidin fusions of atarget protein can also be utilized in pharmokinetic, efficacy or otherstandard assay formats designed to test preclinical samples or clinicalpatient samples for the presence of the therapeutic antibody in blood,urine, or other tissue samples. Avidin fusion protein partners can beprepared as full-length or truncated sequences, specific isolatedstructural domains, or as chimeric sequences with other homologs of thefusion partner from other species.

Avidin fusion proteins can be expressed using any standard means ofintroducing genes into cells, as described herein and known in the art.The proteins can be expressed in, for example, 293 or CHO cells bytransfecting the cells with an avidin fusion construct in a solution oflipids, such as in Lipofectamine (Invitrogen, Carlsbad, Calif.).

Conditioned media and/or cell lysates from cells expressing the fusionproteins can be collected and applied to an assay substrate, such asbiotin-coated polystyrene beads or biotin-coated ELISA plates.Collecting the conditioned media and/or cell lysate can be conducted ata time point that allows for optimum expression of the fusion protein.The time point can be determined experimentally by those skilled in theart, but is usually about 48 hours post-transfection. Fusion proteinscan also be analyzed at the cell membrane or intracellularly forexpression and functionality in binding known ligands, receptors, orantibodies.

Avidin fusion proteins of the invention can be analyzed by any known orpreviously characterized method that utilizes biotin-avidininteractions. Such methods include, but are not limited to, flowcytometry and fluorescent imaging/microscopy. For example, avidinfusions expressed in media or cell lysates can be applied tobiotin-coated beads and stained with a fluorescently tagged anti-avidinantibody to indicate expression level. Also, fluorescent antibodies canbe applied that recognize the specific fusion protein partner in amulticolorimetric assay format. Additionally, unlabeled antibodiesspecific for the fusion protein partner can be applied simultaneouslywith fluorescently tagged antibodies in a competition assay.

In certain embodiments, the invention provides methods for mappingepitopes using avidin fusion proteins. An example of an epitope mappingmethod of the invention is provided below with respect to mappingepitopes for anti-IL-1R1 antibodies. However, one of skill in the artwill recognize that such methods can be readily applied to mappingepitopes for any antibody and is not limited to anti-IL-1R1 antibodies.For example, cDNA encoding chicken avidin (with endogenous signalsequence) can be joined with the 5′ end of cDNAs encoding a protein ofinterest (i.e. a protein that is recognized by antibodies for whichdetermining an epitope is desired) fused to a FLAG-tag sequence at the3′ end. The FLAG-tagged fusion genes can be assembled in an expressionvector using conventional molecular techniques. A panel of mutantavidin-FLAG tagged proteins in which certain amino acids have beensubstituted (e.g., with corresponding amino acid residues from anotheranimal species) can be generated using conventional techniques. Themutant and wild type proteins can be expressed in host cells and bindingof the wild-type or mutant proteins with an antibody of interest can bedetected using, for example, Western blot analysis or bead-based bindingassays as described herein. Thus, an epitope can be defined bydetermining which substitutions in the mutant proteins destroy bindingto the antibody of interest.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting the invention.

Example 1 Production of Human Monoclonal Antibodies AgainstInterleukin-1 Receptor Type I (IL-1R1)

Transgenic HuMab Mice

Fully human monoclonal antibodies to IL-1 receptor type I (IL-1R1) wereprepared using the HCo7 strain of transgenic mice, which expresses humanantibody genes. In each of these mouse strains, the endogenous mousekappa light chain gene has been homozygously disrupted as described inChen et al. (1993, EMBO J. 12:811-820), and the endogenous mouse heavychain gene has been homozygously disrupted as described in Example 1 ofInternational Patent Application Publication No. WO 01/09187(incorporated by reference). Each of these mouse strains carries a humankappa light chain transgene, KCo5, as described in Fishwild et al.(1996, Nature Biotechnology 14:845-851). The HCo7 strain carries theHCo7 human heavy chain transgene as described in U.S. Pat. Nos.5,545,806; 5,625,825; and 5,545,807 (incorporated by reference). TheHCo7 strain is referred to herein as HuMab mice.

HuMab Immunizations

To generate fully human monoclonal antibodies to IL-1R1, HuMab mice wereimmunized with purified recombinant IL-1R derived from insect ormammalian cells (for example, CHO cells) as antigen. Generalimmunization schemes for HuMab mice are described in Lonberg et al.(1994, Nature 368:856-859; Fishwild et al., supra; and InternationalPatent Application Publication No. WO 98/24884, the teachings of each ofwhich are incorporated by reference). Mice were 6-16 weeks of age uponthe first infusion of antigen. A purified recombinant preparation (25-50μg) of IL-1R1 antigen (e.g., purified from transfected insect ormammalian cells expressing IL-1R1) was used to immunize the HuMab miceintraperitoneally (IP) or subcutaneously (Sc).

Immunizations of HuMab transgenic mice were achieved using antigen incomplete Freund's adjuvant and two injections, followed by 2-4 weeks IPimmunization (up to a total of 11 immunizations) with the antigen inincomplete Freund's adjuvant. Several dozen mice were immunized for eachantigen. A total of 149 mice of the HCo7 strain were immunized withIL-1R1. The immune response was monitored by retroorbital bleeds.

To select HuMab mice producing antibodies that bound IL-1R1, sera fromimmunized mice were tested by ELISA as described by Fishwild et al.,supra. Briefly, microtiter plates were coated with purified recombinantIL-1R1 from insect or mammalian cells at 1-2 μg/mL in PBS and 50 μL/wellincubated at 4° C. overnight, then blocked with 200 μL/well of 5%chicken serum in PBS/Tween (0.05%). Dilutions of plasma fromIL-1R1-immunized mice were added to each well and incubated for 1-2hours at ambient temperature. The plates were washed with PBS/Tween andthen incubated with a goat-anti-human IgG Fc-specific polyclonal reagentconjugated to horseradish peroxidase (HRP) for 1 hour at roomtemperature. Plates were washed with PBS/Tween and incubated with a goatanti-human IgG Fc-specific polyclonal reagent conjugated to horseradishperoxidase (HRP) for 1 hour at room temperature. After washing, theplates were developed with ABTS substrate (Sigma Chemical Co., St.Louis, Mo., Catalog No. A-1888, 0.22 mg/mL) and analyzedspectrophotometrically at OD of 415-495. Mice with sufficient titers ofanti-IL-1R1 human immunoglobulin were used to produce monoclonalantibodies as described below.

Generation of Hybridomas Producing Human Monoclonal Antibodies to IL-1R1

Mice were prepared for monoclonal antibody production by boosting withantigen intravenously 2 days before sacrifice, and spleens were removedthereafter. The mouse splenocytes were isolated from the HuMab mice andfused with PEG to a mouse myeloma cell line using standard protocols.Typically, 20-30 fusions for each antigen were performed.

Briefly, single cell suspensions of splenic lymphocytes from immunizedmice were fused to one-fourth the number of P3X63-Ag8.653 nonsecretingmouse myeloma cells (A.T.C.C., Accession No. CRL 1580) or SP2/0nonsecreting mouse myeloma cells (A.T.C.C., CRL 1581) with 50% PEG(Sigma). Cells were plated at approximately 1×10⁵/well in flat bottommicrotiter plates, followed by about a two week incubation in selectivemedium containing 10% fetal bovine serum, 10% P388D1—(A.T.C.C.,Accession No. CRL TIB-63) conditioned medium, 3-5% origen (IGEN) in DMEM(Mediatech, Catalog No. CRL 10013, with high glucose, L-glutamine andsodium pyruvate) plus 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 mg/mLgentamycin and 1×HAT (Sigma, Catalog No. CRL P-7185). After 1-2 weeks,cells were cultured in medium in which the HAT was replaced with HT.

The resulting hybridomas were screened for the production ofantigen-specific antibodies. Individual wells were screened by ELISA(described above) for human anti-IL-1R1 monoclonal IgG antibodies. Onceextensive hybridoma growth occurred, medium was monitored usually after10-14 days. Antibody-secreting hybridomas were replated, screened againand, if still positive for human IgG, anti-IL-1R1 monoclonal antibodieswere subcloned at least twice by limiting dilution. The stable subcloneswere then cultured in vitro to generate small amounts of antibody intissue culture medium for characterization.

Selection of Human Monoclonal Antibodies Binding to IL-1R1

An ELISA assay as described above was used to screen for hybridomas thatshowed positive reactivity with IL-1R1 immunogen. Hybridomas secreting amonoclonal antibody that bound with high avidity to IL-1R1 weresubcloned and further characterized. One clone from each hybridoma,which retained the reactivity of parent cells (as determined by ELISA),was chosen for making a 5-10 vial cell bank stored in liquid nitrogen.

An isotype-specific ELISA was performed to determine the isotype of themonoclonal antibodies produced as disclosed herein. In theseexperiments, microtiter plate wells were coated with 50 μL/well of asolution of 1 μg/mL of mouse anti-human kappa light chain in PBS andincubated at 4° C. overnight. After blocking with 5% chicken serum, theplates were reacted with supernatant from each tested monoclonalantibody and a purified isotype control. Plates were incubated atambient temperature for 1-2 hours. The wells were then reacted witheither human IgG1, IgG2 or IgG4-specific horseradishperoxidase-conjugated goat anti-human polyclonal antisera and plateswere developed and analyzed as described below.

Monoclonal antibodies purified from hybridoma supernatants that showedsignificant binding to IL-1R1 as detected by ELISA were further testedfor biological activity using in vitro binding assays and humanchondrocyte and whole blood cell-based assays. The antibodies thatdisplayed the best activity were designated 15C4, 26F5, 27F2, 24E12, and10H7. The antibodies were subjected to a preliminary epitope sortingexperiment. ELISA plates were coated with human sIL-1R1 (1+2+3 domain),truncated human sIL-1R1 (1+2 domain), rat sIL-1R1, human sIL-1R type II,and ovalbumin (negative control). Antibody binding was detected with ahorseradish peroxidase-conjugated anti-Human Fc antibody (PierceChemical Co., Rockford, Ill.). The results are summarized in Table 2. Acheck mark (✓) in Table 2 represents a positive result for binding; “X”represents a negative result. Antibodies 15C4, 26F5, 27F2 and 24E12 bindonly the IL-1R1 protein that has all three extracellular domains,indicating that the epitopes for each fall within the third domain.Antibody 10H7 binds both the full-length extracellular domain IL-1R1 andalso a truncated protein that has only domains 1 and 2, demonstratingthat the epitope for this antibody lies within either domain 1 or 2.None of the antibodies tested has cross-reactivity with human type IIreceptor or rat IL-1R1.

TABLE 2 OA Hu sIL-1R1 Hu sIL-1R1 Hu sIL-1RII Rat sIL-1R1 Antibody(Negative Control) (1 + 2 + 3 Domain) (1 + 2 Domain) (1 + 2 + 3 Domain)(1 + 2 + 3 Domain) 15C4 X √ X X X 26F5 X √ X X X 27F2 X √ X X X 24E12 X√ X X X 10H7 X √ √ X X

Example 2 In Vitro Inhibition of IL-1 Receptor Type I Complex Formationby Anti-IL-1R1 Antibodies

The ability of the antibodies to inhibit the extracellular bindingevents required for IL-1 signaling was assessed with recombinantproteins in vitro in an assay in which IL-1 binding to IL-1R results information of a high affinity binding site for IL-1 RAcP. The binding ofIL-1RAcP to IL-1-bound IL-1R (referred to as “complex formation”) ismeasured as follows. Recombinant proteins were incubated in bindingassays in microtiter plates in the absence (control) or presence ofantibodies. IC₅₀ values were derived from comparisons of control valuesto values obtained in the presence of antibody at concentrations between10 fM and 1 μM. In brief, the assay was conducted as follows.Biotinylated IL-1R1 and streptavidin-coated beads (Dynal, DynabeadsM-28) were dispensed in microtiter plates. Antibody was then added tothe appropriate wells in a serial dilution covering a broad range ofconcentrations. IL-1β or IL-1α was added at a concentration of 1 nM, andIL1RAcP labeled with ruthenium (prepared with NHS-Tag (IGEN) accordingto IGEN protocols) was added at a final concentration of 5 nM. Afterincubation for 1 hour at room temperature, the binding reaction wasanalyzed with either an ORIGEN™ 1.5 or M8 instrument (IGEN InternationalInc.). IL-1RAcP binding to IL-1 bound IL-1R1 was determined by detectingthe electrochemiluminescence signal associated with the IL-1R1 boundbeads. The reduction of signal resulting from antibody competition ofeither IL-1 or IL-1RAcP binding was calculated as percentage of ECLsignal for maximum binding (no competition).

The inhibition response curve for each antibody in these binding assayswas established and IC₅₀s were derived using PRISM™ software. Theresults for inhibition of IL-1β induced binding events are depicted bythe graph in FIG. 12. The IC₅₀ values for inhibition of complexformation are shown in Table 3 below. Antibodies 15C4, 26F5, 27F2, and24E12 strongly inhibit complex formation. These antibodies are allIL-1R1 third domain binders, as described above. Antibody 10H7 belongsto a class of antibodies that binds to a construct of the IL-1R lackingthe third domain. 10H7 is a less potent inhibitor of IL-1 driven bindingof IL-1RAcP than the third domain binders. Complex formation inhibitionby the antibodies of the invention was compared with inhibition byIL-1ra. The third domain binders demonstrated similar or slightlygreater ability to inhibit complex formation by comparison with IL-1ra.

FIG. 13 depicts the ability of antibody 15C4 to inhibit IL-1R/IL-1α/RAcPcomplex formation. The IC₅₀ for IL-1R1/IL-1α/RAcP complex formation was43 pM.

TABLE 3 Human anti-IL-1R1 15C4 26F5 27F2 24E12 10H7 rII-1ra IC50 96 pM160 pM 333 pM 348 pM 5.3 nM 555 pM 95% Confidence 71 pM to 129 118 pM to219 214 pM to 517 223 pM to 542 3.6 nM to 7.5 414 pM to Limits pM pM pMpM nM 743 pM

Example 3 Anti-IL-1R1 Antibodies Inhibit Binding of IL-1β and IL-1ra toReceptor

The ability of anti-IL-1R1 antibodies to inhibit binding of either IL-1βor IL-1ra to IL-1R1 was assessed in an assay with recombinant proteins.The reaction mixture contained 0.1 mg/mL Dynabeads M-280 Streptavidin(Dynal) and 1 nM biotinylated IL-1R1. Antibodies were added atconcentrations from 320 nM to 0.3 nM. Addition of ruthenium-tagged IL-1β(5 nM) or IL-1ra (1 nM) initiated binding that proceeded for 1 hour atroom temperature. The reaction mixtures were measured as above using anORIGEN™ 1.5 or M8 instrument (IGEN International Inc.). Competition wascalculated as the percentage of ECL signal for maximum binding (nocompetition). Antibodies 15C4, 26F5, and 27F2, the most potentantibodies, block ligand (IL-1β) binding to receptor, but do notsignificantly interfere with the binding of IL-1ra compared with IgGcontrol. In contrast, antibody 24E12 binds receptor but does not blockIL-1β or IL-1ra binding to receptor. Thus, antibody 24E12 represents aunique class of third domain binders distinct from the class representedby 15C4, 26F5, and 27F2. Antibody 10H7 inhibits both IL-1 and IL-1rafrom binding to the receptor. The results are summarized in FIG. 14.

Example 4 Chondrocyte and Human Whole Blood Assays

Primary human chondrocytes (Cell Applications Inc., San Diego, Calif.)were seeded into 96-well plates at a density of 10,000 cells/well inDMEM media containing 1% FBS and 1% Pen Strep (GIBCO). Cells wereallowed to recover overnight before addition of anti-IL1-R1 antibodiesat concentrations ranging from 10 nM to 0.1 pM for 20 minutes. IL-1β wasadded to a concentration of 1 pM (˜EC₅₀) and culture supernatants wereharvested after 16 hours incubation at 37° C. IL-6 levels in thesupernatant were measured using an ELISA (Pierce-Endogen, Rockford,Ill., Cat#EH2IL-65) according to the manufacturer's instructions. Theinhibition response curve for each antibody of the invention in thecell-based assays was established and IC₅₀ values were derived usingPRISM™ software. Antibodies 15C4, 26F5, and 27F2 are potent inhibitorsof IL-1 signaling compared with IL-1ra (FIG. 15A). Antibodies 24E12 and10H7 are markedly less potent than 15C4 and 27F2 (FIG. 15B). The IC₅₀values for inhibition of IL-1β induced IL-6 production humanchondrocytes are shown in Tables 4A and 4B (corresponding to FIGS. 15Aand 15B respectively).

Anti-IL-1R1 monoclonal antibodies 15C4, 26F5, and 27F2 werepre-incubated 40-60 minutes with human whole blood collected from normalvolunteers in sodium heparin vacutainers. The assays were run asfollows: 100 μL freshly isolated blood was aliquoted wells of a 96-wellplate. 50 μL of antibody was added in RPMI medium containing 10% humanAB serum. IL-1β was then added at a concentration of 30 pM (EC₅₀).Culture supernatants were harvested after 18 hours, and IL-6 levels inthe supernatant were measured using an ELISA. As a control, IL-1ra waspre-incubated 40-60 minutes with whole blood and IL-6 production wasmeasured as above. The three anti-IL-1R1 antibodies blocked IL-1activity with potency comparable to that of IL-1ra (FIG. 16). The IC50values for inhibition of IL-1-induced IL-6 production in human wholeblood are shown in Table 5.

TABLE 4A Human anti-IL-1R1 Antibodies 15C4 27F2 26F5 rIL-1ra IC50 16 pM32 pM 26 pM 32 pM 95% 15 pM to 21 pM to 19 pM to 22 pM to Confidence 18pM 49 pM 36 pM 46 pM Limits

TABLE 4B Human anti-IL-1R1 Antibodies 15C4 27F2 10H7 24E12 rIL-ra IC50 7pM 28 pM 7.5 pM NA 20 pM 95% 5.8 pM to 22 pM to 5.6 pM to NA 17 pM toCon- 7.9 pM 35 pM 10 pM 23 pM fidence Limits

TABLE 5 Analysis Donor Parameters 15C4 26F5 27F2 IL-1ra 1047 IC50 126 pM410 pM 249 pM 241 pM 95% 47 pM to 339 pM 213 pM to 790 pM  88 pM to 703pM 124 pM to 471 pM Confidence Limits 1319 IC50 111 pM 174 pM 579 pM 381pM 95% 59 pM to 208 pM  60 pM to 501 pM 249 pM to 1.3 pM  167 pM to 875pM Confidence Limits Composite IC50 126 pM 372 pM 387 pM 264 pM (PooledData) 95% 62 pM to 255 pM 187 pM to 739 pM 202 pM to 748 pM 134 pM to517 pM Confidence Limits

Example 5 Mutagenesis and Epitope Mapping

Site directed mutagenesis (Altered Sites® In Vitro Mutagenesis System,Promega, Madison Wis.) of IL-1R1 was used to prepare a panel of mutantproteins (“muteins”) in which rat amino acid residues were substitutedfor the corresponding human sequence. Fifteen different mutated plasmidswere constructed (see numbered bars in FIG. 17). Plasmids encoding thesesubstituted proteins and the parental IL-1R1 were transientlytransfected in CHO cells. Mock transfectants were generated as negativecontrols. Conditioned medium (CM) from these cells was concentrated˜20-fold using Centriprep 10 concentration columns (Amicon). Expressionof the muteins was assessed by SDS-PAGE and Western blotting. Thirteenmutant proteins were expressed at levels that allowed evaluation ofantibody binding. The proteins were loaded onto a gel, electrophoresedand transferred to membranes. The membranes were blocked in 1% milk inPBS, 0.1% Tween-20 and then incubated for 1 hour at room temperaturewith anti-IL-1R antibodies 15C4, 27F2, or 24E12 at 0.5 ug/mL in PBS,0.1% Tween-20. After washing, membranes were incubated with goatanti-human IgG-Fc-HRP. Signal was detected using chemiluminescence (ECL)substrate (Pierce Chemical Co., Rockford, Ill.). Human specificsequences critical for antibody binding were identified as those thatwhen substituted with rat sequences reduced or eliminated ECL signal.15C4 recognition of mutants 1, 2, 4 and 10 was impaired when compared to24E12 (FIG. 18, top panel). Similarly, 27F2 binding to mutants 1, 2 and4 was impaired (FIG. 18, middle panel). 24E12 had no significant bindingto mutants 12, 13, 14 and 15 (FIG. 18, bottom panel).

Isolation and characterization of human anti-IL-1R1 antibodies hasidentified three distinct classes of competitive antibodies (FIG. 19).The strongest inhibitors of IL-1 biological activity, as demonstrated bycell-based bioassays, are those antibodies that bind the third domain ofIL-1R1 and prevent IL-1-β association. Epitope mapping experiments usinga panel of third domain mutant proteins has demonstrated that this classof antibodies, which includes 15C4, 27F2 and 26F5, shares an overlappingbut not identical, conformational epitope. FIGS. 20 and 21 illustratethe position of 15C4 epitopes on the third domain of the IL-1 receptor,in a ribbon diagram of IL-1ra bound IL-1 receptor (Schreuder et al.,1997, Nature 386:194-200). The IL-1 receptor residues that definebinding of the most potent class of antibodies are illustrated in gray.These antibodies have demonstrated superior potency, and thus theseepitopes define binding sites for antibodies of a superior class. The15C4 and 27F2 binding sites are overlapping but not identical, asdetermined by the mutational analysis of the 15 different sites withinIL-1R1 described above. The sites are depicted in FIG. 17 as numberedbars above the protein sequence. Critical sites of interaction appear tobe within mutations at sites 1 (LSDIA; SEQ ID NO: 41), 2 (VIDE; SEQ IDNO: 42), 4 (YSV) and 10 (TCFA; SEQ ID NO: 43). 15C4 and 27F2 bindingsites are comprised within sites 1 and 2, since substitution of the ratresidues for the human residues in either site abolishes binding. 27F2differs from 15C4 in that changes in site 4 completely abolish itsbinding, whereas 15C4 binding is reduced but not completely eliminated.Mutation 10 also reduces 15C4 binding, but 27F2 has no obviousinteraction with this site. Examination of the crystal structure revealsthat these residues define a face of the third domain that is orientedtowards the space occupied by bound ligand (FIGS. 20 and 21) (Vigers etal., 1997, Nature 386:190-194).

The second class of antibodies identified, represented by 10H7, does notrequire the third domain for binding, and unlike the preferred class,inhibits IL-1ra binding. This class is active in bioassays, but is lesspotent than the preferred class.

In contrast to the strong inhibition of IL-1 bioassays with thepreferred class of antibodies, 24E12 is an ineffective inhibitor inbioassays. Antibody 24E12 inhibits the binding of IL-1RAcP withIL-1-bound IL-1R. The epitope for this class of antibodies, defined bymutants 12, 13, 14 and 15, is proximal to the transmembrane domain ofIL-1R1, and is in a region not directly involved in either IL-1 orIL-1ra binding (FIG. 22).

Example 6 Cloning the Anti-IL-1R1 Antibody Heavy and Light Chains

Cloning of the anti-IL-1R1 15C4 MAb Light Chain

The light chains for three hybridomas expressing αIL-1R1 bindingmonoclonal antibodies, 15C4, 27F2, and 26F5 were cloned into themammalian cell expression vector pDSRα19 (see International Application,Publication No. WO 90/14363, which is herein incorporated by referencefor any purpose). The construction of the plasmid encoding the 15C4kappa light chain is explicitly described herein; cloning of the otherlight chain species was performed using similar procedures. The αIL-1R1kappa light chain variable region was obtained using polymerase chainreaction (PCR) amplification methods from first strand cDNA preparedfrom αIL-1R1 hybridoma 15C4 total RNA prepared using TRIzol® reagent(Invitrogen). First strand cDNA was synthesized using a random primerwith an extension adapter (5′-GGC CGG ATA GGC CTC CAN NNN NNT-3′; SEQ IDNO: 44) and 5′ RACE (rapid amplification of cDNA ends) was performedusing the GeneRacer™ Kit (Invitrogen). For the complete light chain, theforward primer was the GeneRacer™ nested primer (5′ GGA CAC TGA CAT GGACTG AAG GAG TA-3′; SEQ ID NO: 45) and the reverse primer was 5′-GGG GTCAGG CTG GAA CTG AGG-3′ (SEQ ID NO: 46). The RACE products were clonedinto pCR4-TOPO (Invitrogen) and the DNA sequences were determined. The15C4 kappa chain consensus DNA sequence was used to design primers forfull-length antibody chain PCR amplification. The 5′ kappa PCR primerencoded the amino terminus of the signal sequence, an XbaI restrictionenzyme site, and an optimized Kozak sequence (5′-CAG CAG AAG CTT CTA GACCAC CAT GTC GCC ATC ACA ACT CAT TGG G-3′; SEQ ID NO: 47). The 3′ primerencoded the carboxyl terminus and termination codon, as well as a SalIrestriction site (5′-CTT GTC GAC TCA ACA CTC TCC CCT GTT GAA GCT C-3′;SEQ ID NO: 48).

5′ αIL-1R1 15C4 kappa primer (SEQ ID NO: 47): (SEQ ID NO: 49)5′-CAG CAG AAG CTT CTA GAC CAC CAT GTC GCC                     XbaI   Kozak M   S   P    ATC ACA ACT CAT TGG G-3′  S   Q   L   I   G 3′ αIL-1R1 15C4 kappa primer (SEQ ID NO: 48):(SEQ ID NO: 50) 5′-CTT GTC GAC TCA ACA CTC TCC CCT GTT GAA        SalI    *   C   E   G   R   N   F GCT C-3′  S

The full-length αIL-1R115C4 kappa chain clone was obtained using a pCR4:15C4 kappa clone by PCR amplification with the 5′ and 3′ αIL-1R115C4kappa primers. The PCR reaction generated a 733 base pair productencoding the 233 amino acids residues (including the 19 amino acid kappachain signal sequence) of the αIL-1R1 15C4 kappa chain. The PCR productwas purified using a QIAquick PCR Purification kit (Qiagen Cat. No.28104), cut with XbaI and SalI, gel isolated and purified using aQIAquick Gel Extraction kit (Qiagen Cat. No. 28704). This PCR fragmentcontaining the complete αIL-1R1 15C4 kappa chain was then ligated intothe mammalian expression vector pDSRα19. The 15C4 kappa chain expressionclone was DNA sequenced to confirm that it encoded the same peptide thatwas identified in the 15C4 hybridoma. The final expression vector,pDSRα19:15C4 kappa is 5468 base pairs and contains the seven functionalregions described in Table 6.

TABLE 6 Plasmid Base Pair Number:  2 to 881 A transcriptiontermination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin et al., 1983, NucleicAcids Res. 11: 6873-82; Genbank Accession Number X00004)  882 to 2027 Amouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFRtranscription termination/polyadenylation signals (Gasser et al, 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19:355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGrogan etal., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequencescontaining the ampicillin resistance marker gene and the origin forreplication of the plasmid in E. coli (Genbank Accession Number J01749)3949 to 4292 An SV40 early promoter, enhancer and origin of replication(Takebe et al., 1988, Mol. Cell Biol. 8: 466-72, Genbank AccessionNumber J02400) 4299 to 4565 A translational enhancer element from theHTLV-1 LTR domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3618-22, Genbank Accession Number J02029) 4574 to 4730 An intron fromthe SV40 16S, 19S splice donor/acceptor signals (Okayama and Berg, 1983,Mol. Cell Biol. 3: 280-9, Genbank Accession Number J02400) 4755 to 5468The 15C4 kappa light chain cDNA between the Xba1 and Sal1 sitesConstruction of pDSR19:hIgG1C_(H)

A pDSRα19:rat variable region/human constant region IgG1 (rVh/hCh1) MAbexpression plasmid was constructed as the result of a three-pieceligation of XbaI and BsmBI terminated rat antibody variable region PCRproduct, the human IgG1 constant region (C_(H1), hinge, C_(H2) andC_(H3) domains) derived by Sail cleavage and gel isolation of the BsmBIand SalI fragment from the linear plasmid pDSRα19:hIgG1 C_(H) (HindIIIand BsmBI ends) and a linearized pDSRα19 with XbaI and SalI ends (seeco-owned and co-pending U.S. Provisional Patent Application No.60/370,407, filed Apr. 5, 2002, “Human Anti-OPGL Neutralizing AntibodiesAs Selective OPGL Pathway Inhibitors,” incorporated by reference). Thefinal expression vector, pDSRα19:rat variable region/human constantregion IgG1 (rVh/hCh1), is 6158 base pairs and contains the 7 functionalregions described in Table 7.

TABLE 7 Plasmid Base Pair Number:   2 to 881A transcription termination/polyadenylation signal from theα-subunit of the bovine pituitary glycoprotein hormone (α-FSH)(Goodwin et al., 1983, Nucleic Acids Res. 11: 6873-82; GenbankAccession Number X00004) 882 to 2027A mouse dihydrofolate reductase (DHFR) minigene containing theendogenous mouse DHFR promoter, the cDNA coding sequences, andthe DHFR transcription termination/polyadenylation signals (Gasseret al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg etal., 1980, Cell 19: 355-64; Setzer et al., 1982, J. Biol. Chem.257: 5143-7; McGrogan et al., 1985, J. Biol. Chem. 260: 2307-14)2031 to 3947pBR322 sequences containing the ampicillin resistance marker geneand the origin for replication of the plasmid in E. coli (GenbankAccession Number J01749) 3949 to 4292An SV40 early promoter, enhancer and origin of replication (Takebeet al., 1988, Mol. Cell Biol. 8: 466-72, Genbank Accession NumberJ02400) 4299 to 4565A translational enhancer element from the HTLV-1 LTR domain(Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 3618-22,Genbank Accession Number J02029) 4574 to 4730An intron from the SV40 16S, 19S splice donor/acceptor signals(Okayama and Berg, 1983. Mol. Cell Biol. 3: 280-9, Genbank AccessionNumber J02400) 4755 to 6158The rVh/hCh1 heavy chain cDNA between the Xba1 and Sal1 sites.This heavy chain fragment sequence is shown below (SEQ ID NO: 51)with the sequences of the restriction sites underlined:  XbaI TCTAG ACCACCATGG ACATCAGGCT CAGCTTAGTT TTCCTTGTCCTTTTCATAAA AGGTGTCCAG TGTGAGGTAG AACTGGTGGAGTCTGGGGGC GGCTTAGTAC AACCTGGAAG GTCCATGACACTCTCCTGTG CAGCCTCGGG ATTCACTTTC AGAACCTATG GCATGGCCTGGGTCCGCCAG GCCCCAACGA AGGGTCTGGA GTGGGTCTCATCAATTACTG CTAGTGGTGG TACCACCTAC TATCGAGACT CCGTGAAGGGCCGCTTCACT ATTTTTAGGG ATAATGCAAA AAGTACCCTA TACCTGCAGATGGACAGTCC GAGGTCTGAG GACACGGCCA CTTATTTCTG TACATCAATT                                   BsmB1TCGGAATACT GGGGCCACGG AGTCATGGTC ACCGTCTCTAGTGCCTCCACCAAGGGCCCA TCGGTCTTCC CCCTGGCACC CTCCTCCAAGAGCACCTCTGGGGGCACAGC GGCCCTGGGC TGCCTGGTCA AGGACTACTTCCCCGAACCG GTGACGGTGT CGTGGAACTC AGGCGCCCTGACCAGCGGCG TGCACACCTT CCCGGCTGTC CTACAGTCCT CAGGACTCTACTCCCTCAGC AGCGTGGTGACCGTGCCCTC CAGCAGCTTG GGCACCCAGACCTACATCG CAACGTGAATCACAAGCCCA GCAACACCAAGGTGGACAAG AAAGTTGAGC CCAAATCTTG TGACAAAACTCACACATGCC CACCGTGCCC AGCACCTGAA CTCCTGGGGGGACCGTCAGT CTTCCTCTTC CCCCCAAAAC CCAAGGACAC CCTCATGATCTCCCGGACCC CTGAGGTCAC ATGCGTGGTG GTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACT GGTACGTGGACGGCGTGGAG GTGCATAATG CCAAGACAAA GCCGCGGGAGGAGCAGTACA ACAGCACGTA CCGTGTGGTC AGCGTCCTCACCGTCCTGCA CCAGGACTGG CTGAATGGCAAGGAGTACAAGTGCAAGGTC TCCAACAAAG CCCTCCCAGCCCCCATCGAG AAAACCATCTCCAAAGCCAA AGGGCAGCCCCGAGAACCAC AGGTGTACAC CCTGCCCCCA TCCGGGATGAGCTGACCAA GAACCAGGTC AGCCTGACCT GCCTGGTCAAAGGCTTCTAT CCCAGCGACA TCGCCGTGGA GTGGGAGAGCAATGGGCAGCCGGAGAACAA CTACAAGACC ACGCCTCCCGTGCTGGACTC CGACGGCTCC TTCTTCCTCT ATAGCAAGCT CACCGTGGACAAGAGCAGGT GGCAGCAGGG GAACGTCTTC TCATGCTCCGTGATGCATGA GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTG TCTCCGGGTA         SalI AATGATAAGT CGAC

The linear plasmid pDSRα19:hIgG1C_(H) was prepared by digesting thepDSR19:rat variable region/human constant region IgG1 plasmid with therestriction enzymes XbaI and BsmBI to remove the rat variable region andpurified using a QIAquick Gel Extraction kit. The linear plasmidpDSRα19:hIgG1C_(H) containing the 1 kbp human IgG constant region domainwas used to accept hybridoma derived αIL-1R antibody variable regions.

Cloning of the Anti-IL1-R1 15C4 MAb Heavy Chain

The heavy chains for ten hybridomas expressing αIL1-R1 bindingmonoclonal antibodies, 15C4, 27F2, and 26F5 were cloned into themammalian cell expression vector pDSRα19. The construction of theplasmid encoding the 15C4 heavy chain is explicitly described; cloningof the other heavy chain species was performed using similar procedures.The αIL-1R1 15C4 heavy chain variable region was obtained using PCRamplification methods from first strand cDNA prepared from αIL1-R1hybridoma 15C4 total RNA prepared using TRIzol® reagent. First strandcDNA was synthesized using a random primer with an extension adapter(5′-GGC CGG ATA GGC CTC CAN NNN NNT-3′; SEQ ID NO: 44) and a 5′ RACE(rapid amplification of cDNA ends) was performed using the GeneRacer™Kit. For the partial length heavy chain, the forward primer was theGeneRacer™ nested primer (5′ GGA CAC TGA CAT GGA CTG AAG GAG TA-3′; SEQID NO: 45) and the reverse primer was 5′-TGA GGA CGC TGA CCA CAC G-3′(SEQ ID NO 52.). The RACE products were cloned into pCR4-TOPO and theDNA sequences were determined. The 15C4 heavy chain variable regionconsensus DNA sequence was used to design primers for the heavy chainvariable region PCR amplification. The 5′ heavy chain PCR primer encodedthe amino terminus of the signal sequence, an XbaI restriction enzymesite, and an optimized Kozak sequence (5′-CAG CAG AAG CTT CTA GAC CACCAT GGG GTC AAC CGC CAT CCT CG-3′; SEQ ID NO: 53). The 3′ primer encodedthe carboxyl end of the variable region, including a naturally occurringsense strand BsnmBI site (5′-GTG GAG GCA CTA GAG ACG GTG ACC AGG GTTCC-3′; SEQ ID NO: 54).

5′αIL-1R1 15C4 heavy chain primer (SEQ ID NO: 53): (SEQ ID NO: 55)5′-CAG CAG AAG CTT CTA GAC CAC C ATG GGG TCA                   XbaI   Kozak   M   G   S ACC GCC ATC CTCG-3′  T   A   I   L 3′αIL-1R1 15C4 heavy chain primer (SEQ ID NO: 54):(SEQ ID NO: 56) 5′-GTG GAG GCA CTA GAG ACG GTG ACC AGG GTT     T   S   A   S   S   V   T   V   L   T                        BsmBICC-3'  G     Construction of the Anti-IL1-R1 IgG1 Heavy Chain Expression Clone

The full-length αIL-1R1 15C4 heavy chain clone was obtained from apCR4:15C4 heavy chain clone by PCR amplification with the 5′ and 3′αIL-1R1 15C4 heavy chain primers. The PCR reaction generated a 442 basepair product encoding the 137 amino acids residues (including the 19amino acid heavy chain signal sequence) of the αIL-1R1 15C4 heavy chainvariable region. The PCR product was purified using a QIAquick PCRPurification kit and then digested with XbaI and BsmBI, gel isolated andpurified using a QIAquick Gel Extraction kit. This fragment containingthe complete αIL-R1 15C4 heavy chain variable region was then ligatedinto the mammalian expression vector pDSRα19:hIgG1C_(H). The 15C4 heavychain IgG1 expression clone was DNA sequenced to confirm that it encodedthe same heavy chain variable region peptide that was identified in the15C4 hybridoma. The final expression vector, pDSRα19:15C4 IgG1 heavychain was 6173 base pairs and contains the seven functional regionsdescribed in Table 8.

TABLE 8 Plasmid Base Pair Number:  2 to 881 A transcriptiontermination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin et al., 1983, NucleicAcids Res. 11: 6873-82; Genbank Accession Number X00004)  882 to 2027 Amouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFRtranscription termination/polyadenylation signals (Gasser et al., 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19:355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGrogan etal., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequencescontaining the ampicillin resistance marker gene and the origin forreplication of the plasmid in E. coli (Genbank Accession Number J01749)3949 to 4292 An SV40 early promoter, enhancer and origin of replication(Takebe et al., 1988, Mol. Cell Biol. 8: 466-72, Genbank AccessionNumber J02400) 4299 to 4565 A translational enhancer element from theHTLV-1 LTR domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3618-22, Genbank Accession Number J02029) 4574 to 4730 An intron fromthe SV40 16S, 19S splice donor/acceptor signals (Okayama and Berg, 1983.Mol. Cell Biol. 3: 280-9, Genbank Accession Number J02400) 4755 to 6173The 15C4 heavy chain IgG1 cDNA between the Xba1 and Sal1 sitesConstruction of pDSR19:hIgG2C_(H)

A pDSRα19:human variable region/human constant region IgG2 (hVh/hCh2)MAb expression plasmid was constructed as the result of a three-pieceligation of XbaI and BsmBI terminated human antibody variable region PCRproduct, a human IgG2 constant region (C_(H1), hinge, C_(H2) and C_(H3)domains) PCR product with BsmBI and SalI ends and a linearized pDSRa19with XbaI and Sail ends. The final expression vector, pDSRα19:humanvariable region/human constant region IgG1 (hVh/hCh2) (see co-owned andco-pending U.S. Provisional Patent Application No. 60/370,407, filedApr. 5, 2002, “Human Anti-OPGL Neutralizing Antibodies As Selective OPGLPathway Inhibitors”), is 6164 base pairs and contains the 7 functionalregions described in Table 9.

TABLE 9 Plasmid Base Pair Number:   2 to 881A transcription termination/polyadenylation signal from the α-subunit of the bovine pituitary glycoprotein hormone (α-FSH) (Goodwin et al.,1983, Nucleic Acids Res. 11: 6873-82; Genbank Accession Number X00004)882 to 2027A mouse dihydrofolate reductase (DHFR) minigene containing theendogenous mouse DHFR promoter, the cDNA coding sequences, andthe DHFR transcription termination/polyadenylation signals (Gasser etal., 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al.,1980, Cell 19: 355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7;McGrogan et al., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequences containing the ampicillin resistance marker gene andthe origin for replication of the plasmid in E. coli (Genbank AccessionNumber J01749) 3949 to 4292 An SV40 early promoter, enhancer and origin of replication (Takebe etal., 1988, Mol. Cell Biol. 8: 466-72, Genbank Accession Number J02400)4299 to 4565 A translational enhancer element from the HTLV-1 LTR domain(Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 3618-22, GenbankAccession Number J02029) 4574 to 4730 An intron from the SV40 16S, 19S splice donor/acceptor signals(Okayama and Berg, 1983. Mol. Cell Biol. 3: 280-9, Genbank AccessionNumber J02400) 4755 to 6164 The hVh/hCh2 heavy chain cDNA between the Xba1 and Sal1 sites. Thesequence of this heavy chain fragment appears below (SEQ ID NO: 57)with the restriction sites underlined: XbaITCTAGA CCACCATGGA CATGAGGGTC CCCGCTCAGC TCCTGGGGCTCCTGCTATTG TGGTTGAGAG GTGCCAGATG TGAGGTCCAGCTGGTGCAGTCTGGGGGAGG CTTGGTACAT CCTGGGGGGT CCCTGAGACTCTCCTGTGCAGGCTCTGGAT TCACCTTCAG TGGCCATGCT TTGCACTGGGTTCGCCAGGCTCCAGGAAAA GGTCTGGAGT GGGTATCAGG TATTGGTACTCATGGTGGGACATACTATGC AGACTCCGTG AAGGGCCGAT TCACCATCTCCAGAGACAATGCCAAGAACT CCTTGTTTCT TCAAATGAAC AGCCTGAGCGCCGAGGACATGGCTGTGTAT TACTGTACAA GAAGAAACTG                                           BsmB1GGGACAATTT GACTACTGGGGCCAGGGAAC CCTGGTCACC GTCTCTAGTGCCTCCACCAA GGGCCCATCGGTCTTCCCCC TGGCGCCCTG CTCCAGGAGCACCTCCGAGA GCACAGCGGCCCTGGGCTGC CTGGTCAAGG ACTACTTCCCCGAACCGGTG ACGGTGTCGTGGAACTCAGG CGCTCTGACCAGCGGCGTGC ACACCTTCCC AGCTGTCCTACAGTCCTCAG GACTCTACTCCCTCAGCAGC GTGGTGACCG TGCCCTCCAGCAACTTCGGC ACCCAGACCTACACCTGCAA CGTAGATCAC AAGCCCAGCAACACCAAGGTGGACAAGACA GTTGAGCGCA AATGTTGTGTCGAGTGCCCACCGTGCCCAG CACCACCTGT GGCAGGACCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAG GACACCCTCA TGATCTCCCG GACCCCTGAGGTCACGTGCGTGGTGGTGGA CGTGAGCCAC GAAGACCCCGAGGTCCAGTT CAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAG ACAAAGCCAC GGGAGGAGCAGTTCAACAGCACGTTCCGTG TGGTCAGCGT CCTCACCGTT GTGCACCAGGACTGGCTGAACGGCAAGGAG TACAAGTGCA AGGTCTCCAACAAAGGCCTCCCAGCCCCCA TCGAGAAAAC CATCTCCAAAACCAAAGGGC AGCCCCGAGAACCACAGGTG TACACCCTGCCCCCATCCCG GGAGGAGATG ACCAAGAACCAGGTCAGCCTGACCTGCCTG GTCAAAGGCT TCTACCCCAG CGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAG AACAACTACAAGACCACACCTCCCATGCTG GACTCCGACG GCTCCTTCTT CCTCTACAGCAAGCTCACCGTGGACAAGAG CAGGTGGCAG CAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTC TGCACAACCA CTACACGCAG                                    SalIAAGAGCCTCT CCCTGTCTCCGGGTAAATGA TAAGTCGAC

The linear plasmid pDSRα19:hIgG2C_(H) was prepared by digesting thepDSR19:human variable region/human constant region IgG2 plasmid with therestriction enzymes XbaI and BsmBI to remove the human variable regionand purified using a QIAquick Gel Extraction kit. The linear plasmidpDSRα19:hIgG2C_(H) containing the 1 kbp human IgG2 constant regiondomain was used to accept hybridoma derived αIL-1R antibody variableregions.

Construction of the Anti-IL1-RI IgG2 Heavy Chain Expression Clone

The αIL-1R1 15C4 heavy chain variable region fragment, described above,was ligated into the mammalian expression vector pDSRα19:hIgG2C_(H). The15C4 heavy chain IgG2 expression clone was DNA sequenced to confirm thatit encoded the same heavy chain variable region peptide that wasidentified in the 15C4 hybridoma. The final expression vector,pDSRα19:15C4 IgG2 heavy chain was 6161 base pairs and contains the sevenfunctional regions described in Table 10.

TABLE 10 Plasmid Base Pair Number:  2 to 881 A transcriptiontermination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin et al., 1983, NucleicAcids Res. 11: 6873-82; Genbank Accession Number X00004)  882 to 2027 Amouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFRtranscription termination/polyadenylation signals (Gasser et al., 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19:355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGrogan etal., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequencescontaining the ampicillin resistance marker gene and the origin forreplication of the plasmid in E. coli (Genbank Accession Number J01749)3949 to 4292 An SV40 early promoter, enhancer and origin of replication(Takebe et al., 1988, Mol. Cell Biol. 8: 466-72, Genbank AccessionNumber J02400) 4299 to 4565 A translational enhancer element from theHTLV-1 LTR domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3618-22, Genbank Accession Number J02029) 4574 to 4730 An intron fromthe SV40 16S, 19S splice donor/acceptor signals (Okayama and Berg, 1983.Mol. Cell Biol. 3: 280-9, Genbank Accession Number J02400) 4755 to 6161The 15C4 heavy chain IgG2 cDNA between the Xba1 and Sal1 sitesConstruction of pDSR19:hIgG4C_(H)

A pDSRα19:human variable region/human constant region IgG4 (hVh/hCh4)MAb expression plasmid was constructed as the result of a three-pieceligation of XbaI and BsmBI terminated human antibody variable region PCRproduct, a gel isolated BsmBI and SalI digested human IgG4 constantregion (C_(H1), hinge, C_(H2) and C_(H3) domains) fragment and alinearized pDSRa19 with XbaI and SalI ends. The final expression vector,pDSRα19:human variable region/human constant region IgG4 (hVh/hCh4) (seeco-owned and co-pending U.S. Provisional Patent Application No.60/370,407, filed Apr. 5, 2002, “Human Anti-OPGL Neutralizing AntibodiesAs Selective OPGL Pathway Inhibitors”), is 6167 base pairs and containsthe 7 functional regions described in Table 11.

TABLE 11 Plasmid Base Pair Number:   2 to 881A transcription termination/polyadenylation signal from the α-subunit of the bovine pituitary glycoprotein hormone (α-FSH) (Goodwin et al.,1983, Nucleic Acids Res. 11: 6873-82; Genbank Accession Number X00004) 882 to 2027A mouse dihydrofolate reductase (DHFR) minigene containing theendogenous mouse DHFR promoter, the cDNA coding sequences, andthe DHFR transcription termination/polyadenylation signals (Gasser etal., 1982, Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al.,1980, Cell 19: 355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7;McGrogan et al., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947pBR322 sequences containing the ampicillin resistance marker gene andthe origin for replication of the plasmid in E. coli (Genbank AccessionNumber J01749) 3949 to 4292An SV40 early promoter, enhancer and origin of replication (Takebe etal., 1988, Mol. Cell Biol. 8: 466-72, Genbank Accession Number J02400)4299 to 4565 A translational enhancer element from the HTLV-1 LTR domain(Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 3618-22, GenbankAccession Number J02029) 4574 to 4730An intron from the SV40 16S, 19S splice donor/acceptor signals(Okayama and Berg, 1983. Mol. Cell Biol. 3: 280-9, Genbank AccessionNumber J02400) 4755 to 6167The hVb/hCh4 heavy chain cDNA between the Xba1 and Sal1 sites. Thesequence of this heavy chain fragment appears below (SEQ ID NO: 58)with the restriction sites underlined:  XbaITCT AGACCACCAT GGACATGAGG GTCCCCGCTC AGCTCCTGGGGCTCCTGCTA TTGTGGTTGA GAGGTGCCAG ATGTGAGGTCCAGCTGGTGCAGTCTGGGGG AGGCTTGGTA CATCCTGGGGGGTCCCTGAG ACTCTCCTGTGCAGGCTCTG GATTCACCTT CAGTGGCCATGCTTTGCACT GGGTTCGCCAGGCTCCAGGA AAAGGTCTGG AGTGGGTATCAGGTATTGGT ACTCATGGTGGGACATACTA TGCAGACTCC GTGAAGGGCCGATCCACCAT CTCCAGAGACAATGCCAAGA ACTCCTTGTT TCTTCAAATGAACAGCCTGA GCGCCGAGGACATGGCTGTG TATTACTGTACAAGAAGAAA CTGGGGACAA TTTGACTACTGGGGCCAGGG               BsmB1AACCCTGGTC ACCGTCTCTA GTGCCAGCAC CAAGGGGCCATCCGTCTTCCCCCTGGCGCC CTGCTCCAGG AGCACCTCCGAGAGCACAGCCGCCCTGGGC TGCCTGGTCA AGGACTACTTCCCCGAACCG GTGACGGTGTCGTGGAACTC AGGCGCCCTGACCAGCGGCG TGCACACCTT CCCGGCTGTCCTACAGTCCT CAGGACTCTACTCCCTCAGC AGCGTGGTGA CCGTGCCCTCCAGCAGCTTG GGCACGAAGACCTACACCTG CAACGTAGAT CACAAGCCCAGCAACACCAAGGTGGACAAG AGAGTTGAGT CCAAATATGG TCCCCCATGCCCATCATGCCCAGCACCTGA GTTCCTGGGG GGACCATCAG TCTTCCTGTTCCCCCCAAAACCCAAGGACA CTCTCATGAT CTCCCGGACC CCTGAGGTCACGTGCGTGGTGGTGGACGTG AGCCAGGAAG ACCCCGAGGTCCAGTTCAACTGGTACGTGG ATGGCGTGGA GGTGCATAATGCCAAGACAA AGCCUCUUGAGUAGCAGTTC AACAGCACGTACCGTGTGGT CAGCGTCCTC ACCGTCCTGCACCAGGACTG GCTGAACGGCAAGGAGTACA AGTGCAAGGT CTCCAACAAAGGCCTCCCGTCCTCCATCGA GAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAGCCA CAGGTGTACA CCCTGCCCCCATCCCAGGAG GAGATGACCAAGAACCAGGT CAGCCTGACCTGCCTGGTCA AAGGCTTCTA CCCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCC GTGCTGGACT CCGACGGCTC CTTCTTCCTCTACAGCAGGCTAACCGTGGA CAAGAGCAGG TGGCAGGAGGGGAATGTCTT CTCATGCTCCGTGATGCATG AGGCTCTGCA CAACCACTAC                                      SalIACACAGAAGA GCCTCTCCCTGTCTCTGGGT AAATGATAAG TCGAC

The linear plasmid pDSRα19:hIgG4C_(H) was prepared by digesting thepDSR19:human variable region/human constant region IgG4 plasmid with therestriction enzymes XbaI and BsmBI to remove the human variable regionand purified using a QIAquick Gel Extraction kit. The linear plasmidpDSRα19:hIgG4C_(H) containing the 1 kbp human IgG4 constant regiondomain was used to accept hybridoma derived αIL-1R antibody variableregions.

Construction of the Anti-IL1-RI IgG4 Heavy Chain Expression Clone

The αIL-1R1 15C4 heavy chain variable region fragment, described above,was ligated into the mammalian expression vector pDSRα19:hIgG4C_(H). The15C4 heavy chain IgG4 expression clone was DNA sequenced to confirm thatit encoded the same heavy chain variable region peptide that wasidentified in the 15C4 hybridoma. The final expression vector,pDSRα19:15C4 IgG4 heavy chain was 6164 base pairs and contains the sevenfunctional regions described in Table 12.

TABLE 12 Plasmid Base Pair Number:  2 to 881 A transcriptiontermination/polyadenylation signal from the α-subunit of the bovinepituitary glycoprotein hormone (α-FSH) (Goodwin et al., 1983, NucleicAcids Res. 11: 6873-82; Genbank Accession Number X00004)  882 to 2027 Amouse dihydrofolate reductase (DHFR) minigene containing the endogenousmouse DHFR promoter, the cDNA coding sequences, and the DHFRtranscription termination/polyadenylation signals (Gasser et al., 1982,Proc. Natl. Acad. Sci. U.S.A. 79: 6522-6; Nunberg et al., 1980, Cell 19:355-64; Setzer et al., 1982, J. Biol. Chem. 257: 5143-7; McGrogan etal., 1985, J. Biol. Chem. 260: 2307-14) 2031 to 3947 pBR322 sequencescontaining the ampicillin resistance marker gene and the origin forreplication of the plasmid in E. coli Genbank Accession Number J01749)3949 to 4292 An SV40 early promoter, enhancer and origin of replication(Takebe et al., 1988, Mol. Cell Biol. 8: 466-72, Genbank AccessionNumber J02400) 4299 to 4565 A translational enhancer element from theHTLV-1 LTR domain (Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3618-22, Genbank Accession Number J02029) 4574 to 4730 An intron fromthe SV40 16S, 19S splice donor/acceptor signals (Okayama and Berg, 1983.Mol. Cell Biol. 3: 280-9, Genbank Accession Number 302400) 4755 to 6164The 15C4 heavy chain IgG4 cDNA between the Xba1 and Sal1 sites

Example 7 Expression of Anti-IL-1R1 Antibodies in Chinese Hamster Ovary(CHO) Cells

Recombinant anti-IL-1R1 antibodies are generated in Chinese hamsterovary cells, specifically CHO AM-1/D, as disclosed in U.S. Pat. No.6,210,924 (incorporated by reference). Briefly, the DNA sequencesencoding the complete heavy or light chains of each anti-IL-1R1 antibodyof the invention are cloned into expression vectors. CHO AM-1/D cellsare co-transfected with an expression vector capable of expressing acomplete heavy chain and an expression vector expressing the completelight chain of the appropriate anti-IL-1R1 antibody. For example, togenerate the 26F5 antibody, cells are co-transfected with a vectorcapable of expressing a complete light chain comprising the amino acidsequence as set forth in SEQ ID NO:38 and a vector capable of expressinga complete heavy chain comprising the amino acid sequence set forth inSEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24. To generate the 27F2antibody, cells are co-transfected with a vector capable of expressing acomplete light chain comprising the amino acid sequence as set forth inSEQ ID NO: 38 and a vector capable of expressing a complete heavy chaincomprising the amino acid sequence set forth in SEQ ID NO: 26, SEQ IDNO: 28, or SEQ ID NO: 30. To generate the 15C4 antibody, cells areco-transfected with a vector capable of expressing a complete lightchain comprising the amino acid sequence as set forth in SEQ ID NO: 40and a vector capable of expressing a complete heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 34, or SEQ IDNO: 36. Table 13 summarizes the complete heavy and complete light chainsfor the various IL-1R1 antibodies. The designation “ . . . /IgG_”describes the sequence of the constant region for the particularantibody.

TABLE 13 Heavy Chain Variable Region + Complete Antibody Heavy ChainConstant Region Heavy Chain 26F5/IgG1 SEQ ID NO: 9 + SEQ ID NO: 1 SEQ IDNO: 19 (nucleotide) 26F5/IgG1 SEQ ID NO: 10 + SEQ ID NO: 2 SEQ ID NO: 20(amino acid) 26F5/IgG2 SEQ ID NO: 9 + SEQ ID NO: 5 SEQ ID NO: 21(nucleotide) 26F5/IgG2 SEQ ID NO: 10 + SEQ ID NO: 6 SEQ ID NO: 22 (aminoacid) 26F5/IgG4 SEQ ID NO: 9 + SEQ ID NO: 7 SEQ ID NO: 23 (nucleotide)26F5/IgG4 SEQ ID NO: 10 + SEQ ID NO: 8 SEQ ID NO: 24 (amino acid)27F2/IgG1 SEQ ID NO: 13 + SEQ ID NO: 1 SEQ ID NO: 25 (nucleotide)27F2/IgG1 SEQ ID NO: 14 + SEQ ID NO: 2 SEQ ID NO: 26 (amino acid)27F2/IgG2 SEQ ID NO: 13 + SEQ ID NO: 5 SEQ ID NO: 27 (nucleotide)27F2/IgG2 SEQ ID NO: 14 + SEQ ID NO: 6 SEQ ID NO: 28 (amino acid)27F2/IgG4 SEQ ID NO: 13 + SEQ ID NO: 7 SEQ ID NO: 29 (nucleotide)27F2/IgG4 SEQ ID NO: 14 + SEQ ID NO: 8 SEQ ID NO: 30 (amino acid)15C4/IgG1 SEQ ID NO: 15 + SEQ ID NO: 1 SEQ ID NO: 31 (nucleotide)15C4/IgG1 SEQ ID NO: 16 + SEQ ID NO: 2 SEQ ID NO: 32 (amino acid)15C4/IgG2 SEQ ID NO: 15 + SEQ ID NO: 5 SEQ ID NO: 33 (nucleotide)15C4/IgG2 SEQ ID NO: 16 + SEQ ID NO: 6 SEQ ID NO: 34 (amino acid)15C4/IgG4 SEQ ID NO: 15 + SEQ ID NO: 7 SEQ ID NO: 35 (nucleotide)15C4/IgG4 SEQ ID NO: 16 + SEQ ID NO: 8 SEQ ID NO: 36 (amino acid) LightChain Variable Region + Complete Antibody Light Chain Constant RegionLight Chain 26F5/27F2 SEQ ID NO: 11 + SEQ ID NO: 3 SEQ ID NO: 37(nucleotide) 26F5/27F2 SEQ ID NO: 12 + SEQ ID NO: 4 SEQ ID NO: 38 (aminoacid) 15C4 SEQ ID NO: 17 + SEQ ID NO: 3 SEQ ID NO: 39 (nucleotide) 15C4SEQ ID NO: 18 + SEQ ID NO: 4 SEQ ID NO: 40 (amino acid)

Stable expression of anti-IL-1R1 antibodies is achieved byco-transfecting dihydrofolate reductase deficient (DHFR⁻) CHO AM-1/Dcells with the expression vectors. Transfections are carried out usingstandard techniques (calcium phosphate co-precipitation) and DHFRselection. Transfected colonies are isolated and grown to confluence in24-well plates. Antibodies produced by transfected cells are examinedfor appropriate folding and neutralizing activity. Clones overproducingappropriately folded anti-IL-1R1 antibodies of the IgG1, IgG2, and IgG4isotypes are selected and antibodies are purified as described below.

Example 8 Production of Anti-IL-1R1 Antibody

Anti-IL-1R1 antibodies are produced by expression in a clonal line ofCHO cells. For each production run, cells from a single vial are thawedinto serum-free cell culture media. The cells are grown initially in aT-flask and are serially expanded through a series of spinner flasksuntil sufficient inoculum has been generated to seed a 20 L bioreactor.Following growth for 5-10 days, the culture is then used to inoculate a300 L bioreactor. Following growth for an additional 5-10 days, theculture is used to inoculate a 2000 L bioreactor. Production is carriedout in a 2000 L bioreactor using a fed batch culture, in which anutrient feed containing concentrated media components is added tomaintain cell growth and culture viability. Production lasts forapproximately two weeks during which time anti-IL1-R1 antibody isconstitutively produced by the cells and secreted into the cell culturemedium.

The production reactor is controlled at set pH, temperature, anddissolved oxygen level: pH is controlled by carbon dioxide gas andsodium carbonate addition; dissolved oxygen is controlled by air,nitrogen, and oxygen gas flows.

At the end of production, the cell broth is fed into a disk stackcentrifuge and the culture supernatant is separated from the cells. Theconcentrate is further clarified through a depth filter followed by a0.2 μm filter. The clarified conditioned media is then concentrated bytangential flow ultrafiltration. The conditioned media is concentrated15- to 30-fold. The resulting concentrated conditioned medium is theneither processed through purification or frozen for purification at alater date.

Example 9 Epitope Mapping Using Avidin-Fusion Proteins

To generate avidin-fusion proteins, cDNA encoding chicken avidin (withendogenous signal sequence) was joined with the 5′ end of cDNAs encodingthe mature extracellular domains of human- or cynomolgus IL-1R1 fused toa FLAG-tag sequence at the 3′ end. The FLAG-tagged fusion genes wereassembled in a pALTERMAX vector using conventional molecular techniques.The amino acid sequence of the avidin-human IL-1R1 fusion protein isshown in FIG. 23 (SEQ ID NO: 59). The amino acid sequence of theavidin-cynomolgus IL-1R1 fusion protein is shown in FIG. 24 (SEQ ID NO:60). A panel of mutant avidin-cynoIL-1R1-FLAG proteins in which humanamino acids were substituted for the corresponding cynomolgus residueswas generated using the Altered Sites II Mammalian In Vitro MutagenesisSystem (Promega Corp.). The mutations are illustrated in FIG. 24.

Plasmids encoding the avidin-cynoIL-1R mutant and wild-type proteins aswell as the avidin-huIL-R1-FLAG protein were transiently transfectedinto 293T cells using Cytofectine transfection reagent (Bio-RadLaboratories, Inc.). Mock transfectants were used as negative controls.Anti-huIL-1R1 monoclonal antibody (MAb) binding to these proteins wasevaluated by Western blot and bead-based binding assays usingconditioned medium (CM) harvested from the transfected cells.

For Western blot analysis, CM was diluted 1:3 in non-reducing SDS samplebuffer, boiled for 5-10 minutes and loaded onto 10% Tris-glycine gels.Following SDS-PAGE and Western transfer, the membranes were blocked with3% BSA/11% ovalbumin in PBS/0.1% Tween-20 (PBST) and stained withanti-huIL-1R1MAbs. A goat anti-human IgG-Fc-HRP antibody (PierceChemical Co.) diluted 1:15,000 in PBST was used for secondary detection.Anti-FLAG detection was used to normalize for protein loading. Imagecapture and densitometry were performed using a Fluor Chem 8000 digitalimaging system (Alpha Innotech Corp.). The signal intensities for theanti-huIL-1R1MAbs were normalized against the values for the anti-FLAGantibody to account for variation in protein loading. Antibody bindingwas expressed as a percentage of binding to the avidin-humanIL-1R1-FLAG.

The results of the Western blot are shown in FIG. 25A. FIG. 25B showsthe densitometric analysis of a duplicate set of Western blotexperiments. Human residues critical for antibody binding are those thatrestore signal when substituted into cynoIL-1R1. In general, mutations 1and 2 (illustrated in FIG. 24), alone or in combination, restoredbinding to many of the antibodies (15C4/IgG2, 5B8, 1C2, 24H2, 16E9, 26E4and 20G1) while mutations 10.1 and 10.2 did not. None of theseantibodies bound to wild-type cynoIL-1R1. Two antibodies (27F2 and 19C8)bound consistently to all the mutant proteins as well as to wild-typecynoIL-1R1. This suggested that epitope 4 (residues Y279-V281 ofcynoIL-1R1), identified in the rat/human paralog proteins and unchangedin cynomolgus IL-1R1, was the dominant epitope for these antibodies.Epitope 4 is bold, italicized, and underlined in the amino acid sequenceshown in FIG. 24.

In the multiplexed bead-based binding assays, avidin fusion proteinswere captured by incubation of the CM with biotin-coated fluorescentbeads, one bead set per fusion protein (Beadlyte Multi-Biotin 10plexBead Kit; Upstate Biotechnologies). The beads were washed and pooled inPBST and aliquoted to the wells of a 96-well filter bottom plate(Millipore Corp.). Antibodies (anti-huIL-1R1MAbs or anti-FLAG MAb) wereadded at 25 μg/ml and incubated for 1 hour. The beads were again washedand a mixture of Phycoerythrin-conjugated anti-mouse IgG antibody andanti-human IgG (Fab′)2 was used to detect antibody binding. After a 1hour incubation, the beads were washed and resuspended in PBST. Meanfluorescence intensities (MFI) were measured using a Luminex 100(Luminex Corp). The data were normalized using the MFI values foranti-FLAG MAb binding to account for variation in protein loading.Antibody binding was expressed as a percentage of binding to theavidin-huIL-1R1-FLAG (FIG. 26). The binding pattern of the anti-IL-1R1antibodies to the avidin-cynoIL1R1-FLAG proteins mutated with humanresidues as well as to wild-type cynomolgus and human IL-1R1 proteinswas consistent with the immunoblot analysis shown in FIG. 25.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

We claim:
 1. A method for treating an IL-1 mediated disease selectedfrom the group consisting of: pulmonary disease, chronic obstructivepulmonary disease or COPD, pulmonary alveolar proteinosis, bleomycininduced pneumopathy, pulmonary fibrosis, idiopathic pulmonary fibrosis,radiation induced pulmonary fibrosis, cystic fibrosis, collagenaccumulation in the lungs, acute respiratory distress syndrome or ARDS,broncho-pulmonary dysplasia or BPD, chronic obstructive pulmonarydiseases selected from emphysema and chronic bronchitis, chronicfibrotic lung disease, asbestosis, coal worker's pneumoconiosis,silicosis, bronchioliterans organizing pneumonia, pulmonary sarcoidosis,allergic rhinitis, contact dermatitis, atopic dermatitis, diabetes,inflammatory bowel disease, rheumatoid arthritis, and asthma, comprisingadministering to a subject having said IL-1 mediated disease an antibodyor an antigen binding fragment thereof, that specifically binds humaninterleukin-1 receptor type 1 (IL-1R1), wherein the antibody or antigenbinding fragment comprises: a) i) human heavy chain framework regions, aheavy chain CDR1 region comprising SEQ ID NO: 63, a heavy chain CDR2region comprising SEQ ID NO: 66, and a heavy chain CDR3 regioncomprising SEQ ID NO: 69; and ii) human light chain framework regions, alight chain CDR1 region comprising SEQ ID NO: 71, a light chain CDR2region comprising SEQ ID NO: 73, and a light chain CDR3 regioncomprising SEQ ID NO: 75; b) a heavy chain variable region comprisingSEQ ID NO: 80, and a light chain variable region comprising SEQ ID NO:81; or c) a heavy chain variable region comprising an N-terminal orC-terminal deletion of SEQ ID NO: 80, and a light chain variable regioncomprising an N-terminal or C-terminal deletion of SEQ ID NO: 81,wherein said N-terminal or C-terminal deletion of SEQ ID NO: 80comprises at least SEQ ID NO: 63, SEQ ID NO:66, and SEQ ID NO: 69, andwherein said N-terminal or C-terminal deletion of SEQ ID NO: 81comprises at least SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO:
 75. 2.The method of claim 1, wherein the antibody or antigen binding fragmentcomprises i) human heavy chain framework regions, a heavy chain CDR1region comprising SEQ ID NO: 63, a heavy chain CDR2 region comprisingSEQ ID NO: 66, and a heavy chain CDR3 region comprising SEQ ID NO: 69;and ii) human light chain framework regions, a light chain CDR1 regioncomprising SEQ ID NO: 71, a light chain CDR2 region comprising SEQ IDNO: 73, and a light chain CDR3 region comprising SEQ ID NO:
 75. 3. Themethod of claim 1, wherein the antibody comprises a heavy chain variableregion comprising SEQ ID NO: 80, and a light chain variable regioncomprising SEQ ID NO:
 81. 4. The method of claim 1, wherein the antibodyor antigen binding fragment comprises a heavy chain variable regioncomprising an N-terminal or C-terminal deletion of SEQ ID NO: 80, and alight chain variable region comprising an N-terminal or C-terminaldeletion of SEQ ID NO: 81, wherein said N-terminal or C-terminaldeletion of SEQ ID NO: 80 comprises at least SEQ ID NO: 63, SEQ IDNO:66, and SEQ ID NO: 69, and wherein said N-terminal or C-terminaldeletion of SEQ ID NO: 81 comprises at least SEQ ID NO: 71, SEQ ID NO:73, and SEQ ID NO:
 75. 5. The method of claim 1, wherein the heavy chainand light chain of the antigen binding fragment are connected by aflexible linker to form a single-chain antibody.
 6. The method of claim1, wherein the antigen binding fragment of the antibody is asingle-chain Fv, a Fab, a Fab′ or a (Fab′)₂.
 7. The method of claim 1,wherein the antibody or antigen binding fragment thereof is a fullyhuman antibody or fully human antigen binding fragment.
 8. The method ofclaim 1, wherein the antibody comprises an IgG2 constant region.
 9. Themethod of claim 3, wherein the antibody comprises an IgG2 constantregion.
 10. The method of claim 4, wherein the antibody comprises anIgG2 constant region.
 11. The method of claim 1, wherein the antibody orantigen binding fragment thereof inhibits binding of IL-1β to IL-1R1.12. A method for inhibiting IL-1 signaling in a subject having an IL-1mediated disease selected from the group consisting of: pulmonarydisease, chronic obstructive pulmonary disease or COPD, pulmonaryalveolar proteinosis, bleomycin induced pneumopathy, pulmonary fibrosis,idiopathic pulmonary fibrosis, radiation induced pulmonary fibrosis,cystic fibrosis, collagen accumulation in the lungs, acute respiratorydistress syndrome or ARDS, broncho-pulmonary dysplasia or BPD, chronicobstructive pulmonary diseases selected from emphysema and chronicbronchitis, chronic fibrotic lung disease, asbestosis, coal worker'spneumoconiosis, silicosis, bronchioliterans organizing pneumonia,pulmonary sarcoidosis, allergic rhinitis, contact dermatitis, atopicdermatitis, diabetes, inflammatory bowel disease, rheumatoid arthritis,and asthma, comprising administering to the subject an antibody or anantigen binding fragment thereof, that specifically binds humaninterleukin-1 receptor type 1 (IL-1R1) and inhibits binding of IL-1β toIL-1R1, wherein the antibody or antigen binding fragment comprises: a)i) human heavy chain framework regions, a heavy chain CDR1 regioncomprising SEQ ID NO: 63, a heavy chain CDR2 region comprising SEQ IDNO: 66, and a heavy chain CDR3 region comprising SEQ ID NO: 69; and ii)human light chain framework regions, a light chain CDR1 regioncomprising SEQ ID NO: 71, a light chain CDR2 region comprising SEQ IDNO: 73, and a light chain CDR3 region comprising SEQ ID NO: 75; b) aheavy chain variable region comprising SEQ ID NO: 80, and a light chainvariable region comprising SEQ ID NO: 81; or c) a heavy chain variableregion comprising an N-terminal or C-terminal deletion of SEQ ID NO: 80,and a light chain variable region comprising an N-terminal or C-terminaldeletion of SEQ ID NO: 81, wherein said N-terminal or C-terminaldeletion of SEQ ID NO: 80 comprises at least SEQ ID NO: 63, SEQ IDNO:66, and SEQ ID NO: 69, and wherein said N-terminal or C-terminaldeletion of SEQ ID NO: 81 comprises at least SEQ ID NO: 71, SEQ ID NO:73, and SEQ ID NO:
 75. 13. The method of claim 12, wherein the antibodyor antigen binding fragment comprises i) human heavy chain frameworkregions, a heavy chain CDR1 region comprising SEQ ID NO: 63, a heavychain CDR2 region comprising SEQ ID NO: 66, and a heavy chain CDR3region comprising SEQ ID NO: 69; and ii) human light chain frameworkregions, alight chain CDR1 region comprising SEQ ID NO: 71, a lightchain CDR2 region comprising SEQ ID NO: 73, and a light chain CDR3region comprising SEQ ID NO:
 75. 14. The method of claim 12, wherein theantibody comprises a heavy chain variable region comprising SEQ ID NO:80, and a light chain variable region comprising SEQ ID NO:
 81. 15. Themethod of claim 12, wherein the antibody or antigen binding fragmentcomprises a heavy chain variable region comprising an N-terminal orC-terminal deletion of SEQ ID NO: 80, and a light chain variable regioncomprising an N-terminal or C-terminal deletion of SEQ ID NO: 81,wherein said N-terminal or C-terminal deletion of SEQ ID NO: 80comprises at least SEQ ID NO: 63, SEQ ID NO:66, and SEQ ID NO: 69, andwherein said N-terminal or C-terminal deletion of SEQ ID NO: 81comprises at least SEQ ID NO: 71, SEQ ID NO: 73, and SEQ ID NO:
 75. 16.The method of claim 12, wherein the antigen binding fragment of theantibody is a single-chain Fv, a Fab, a Fab′ or a (Fab′)₂.
 17. Themethod of claim 12, wherein the antibody or antigen binding fragmentthereof is a fully human antibody or fully human antigen bindingfragment.
 18. The method of claim 12, wherein the antibody comprises anIgG2 constant region.
 19. The method of claim 14, wherein the antibodycomprises an IgG2 constant region.
 20. The method of claim 15, whereinthe antibody comprises an IgG2 constant region.
 21. The method of claim12, wherein the antibody or antigen binding fragment thereof does notsignificantly inhibit binding of IL-1ra to IL-1R1.